On Kincaidiana Altman, 1936 and Guestphalinus Michaelsen, 1933 (Annelida, Clitellata, Lumbriculidae), with the descriptions of three new species

Two formerly monotypic lumbriculid genera, Guestphalinus Michaelsen, 1933 and Kincaidiana Altman, 1936, are reviewed using morphological and molecular data, following the discovery of new northwestern, Nearctic species. Several populations of Kincaidiana hexatheca Altman, 1936 were examined, and both morphology and DNA data suggest a single, variable species in Pacifi c drainages extending from northern California through Washington, USA. Specimens of Kincaidiana from the Smith River drainage with a single, median atrium and differing genetically from K. hexatheca are assigned to K. smithi sp. nov. The chaetal morphology of North American Guestphalinus populations is variable, and two basic morphotypes are assigned to G. elephantinus sp. nov. and G. exilis sp. nov. This decision is supported by molecular data. The tree topology, based on the mitochondrial 16S rRNA and Cytochrome Oxidase I (COI), and the nuclear 28S rRNA gene sequences, confi rmed the close phylogenetic relationships among the Nearctic Guestphalinus, Kincaidiana and Uktena Fend, Rodriguez & Lenat, 2015. Probable synapomorphies associating these genera include a fi liform, ringed proboscis, a forward shift of reproductive organs relative to the usual position in the family, and spermathecae in the atrial segment.


Introduction
resorption of reproductive organs, sperm sacs and egg sacs almost empty, spermathecae without darklystaining sperm) are described in some cases.Measurements of chaetae are based on slide-mounted worms, but body lengths and widths were supplemented with measurements obtained from unmounted worms in alcohol; unless otherwise noted, measurements of somatic characters were based on specimens with at least partially-developed reproductive pores.Measurements are given as ranges, or as a range followed by the median value in parentheses.Segment numbers are indicated by Roman numerals; intersegments by Arabic numerals (as 9/10 to indicate the septum between IX and X).To simplify the descriptions, we use the general spatial terms "ental vs ectal" (internal vs body wall) for the orientation of internal organs having external pores.For description of chaetae we have used the terms "proximal/ distal", which refer to the distance to the anchor point in the body wall.
Posterior parts of selected specimens (Table 1) were cut and transferred into 95% ethanol for the molecular analyses; anterior parts were treated following the procedures described above and identifi ed to species level.DNA from tissue of posterior parts was extracted using the DNAeasy Tissue Kit (Qiagen) or the QuickExtract DNA Extraction Solution 1.0 from Epicentre (following instructions from the manufacturer).We used primers LCO1490 and HCO2198 (Folmer et al. 1994) or COI-E (Bely & Wray 2004) for COI, 16SAR-L and 16SBRH (Palumbi et al. 1991) for 16S and 28SC1 and 28SC2 (Dayrat et al. 2001) for the 28S rRNA.Each PCR reaction consisted of 21 μl ddH2O, 1 μl of each primer (10 μM), 2 μl template DNA and Illustra PuReTaq Ready-To-Go PCR Beads (0.2 ml tubes) (GE Healthcare).Conditions for PCR were 95ºC/300s, (95ºC/30-40s, 45-54ºC/30-45s, 72ºC/30-60s)* 35 cycles, 72ºC/480s.PCR products were purifi ed using Exonuclease I (Fermentas) and FastAP Thermosensitive Alkaline Phosphatase (Fermentas) and sequenced at Macrogen Sequencing System, South Korea or at MWG Eurofi ns Operon in Edersberg, Germany.Genious 5.3 (Biomatters Ltd.) was used to obtain consensus sequences from assembled forward and reverse sequences, and to edit them.Alignments were made using Clustal X (Larkin et al. 2007) with default settings and then manually corrected in Geneious if necessary.Reading frame shifts were checked for the coding gene COI.Alignments resulted in 658 bp of COI, 496 bp of 16S and 331 bp of 28S data.Trees were built using Bayesian inference (BA) and maximum likelihood (ML).For Bayesian analysis (BA) we used MrBayes v. 3.2.6 (Ronquist & Huelsenbeck 2003).The best-fi t models were selected using the Akaike information criterion (AIC) in MrModeltest v. 2.3 (Nylander 2004) in conjunction with PAUP* v. 4.0b10 (Swofford 2002).For COI, a partitioned site specifi c rate model was used: we applied the Symmetrical model of sequence evolution (SYM) with a proportion of the sites invariable for the COI 1 st codon position, the Felsenstein model (F81) for the COI 2 nd codon position and the Hasegawa-Kishino-Yano model (HKY) with gamma distributed rates across sites for the COI 3 rd codon position.For 16S, we applied the General Time Reversible model (GTR) with gamma distributed rates across sites.For 28S, the model selected was GTR with a proportion of the sites invariable.The number of generations was set to one million with four parallel chains (three hot and one cold), sample frequency to every 100 th generation, and number of runs to two.The fi rst 2500 samples were discarded as the 'burn-in' of the Markov chain.We checked for stationary and convergence of the chains with TRACER v. 1.6 (Rambaut et al. 2014).
For ML analysis we used the online version of RaxML BlackBox (Stamakis et al. 2008), with 100 bootstrap replicates by using the GTR ϒ model.All model parameters were estimated by the program from its own maximum parsimony starting trees.The alignment was divided into the same 5 partitions as used for the Bayesian analysis.Phylogenetic relationships were considered strongly supported if posterior probabilities (pp) in Bayesian analysis were ≥ 0.95 and/or bootstrap values (bv) in maximum likelihood analysis were ≥ 70% (Hillis & Bull 1993;Alfaro et al. 2003).Uncorrected (p) sequence distances were generated using PAUP*.
Type specimens are deposited in the U.S. National Museum of Natural History, Smithsonian Institution, Washington, D.C., USA (USNM), California Academy of Sciences, Invertebrate Zoology, San Francisco, California, USA (CASIZ) and Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN).FEND S.V. et al., Nearctic Kincaidiana and Guestphalinus Generated sequences are deposited in GenBank.Additional material is retained in the collections of S. Fend, U.S. Geological Survey, Menlo Park, California, USA or P. Rodriguez, University of the Basque Country, Spain.Detailed information on specimens used for molecular analysis, with museum voucher and GenBank numbers, are given in Table 1.Additional site locality data, with geographic coordinates, are given in Appendix 1.

Remarks
The diagnosis in Fend (2009) is modifi ed to include the new species with a single, median male pore and atrium, and spermathecal pores in line with ventral chaetae.New observations of several populations of K. hexatheca (see below) indicate that chaetae in anterior segments may appear as simple bifi ds (as European Journal of Taxonomy 361: 1-46 (2017) in previous descriptions), but typically have more complex structure: a broad proximal tooth, a smaller dorsal tooth, and a thin dorsal keel.Anterior chaetae are rarely simple-pointed.Altman (1936) described K. hexatheca and discussed the relationship of Kincaidiana to another proboscisbearing (Holarctic) genus, Rhynchelmis Hoffmeister, 1843, but made no mention of the European Guestphalinus wiardi (Michaelsen, 1933).Rhynchelmis differs from Kincaidiana, Guestphalinus and Uktena in having spermathecae only in segments anterior to the atria, and the (usually) semiprosoporous atria are (almost always) located in segment X. Kincaidiana freidris Cook, 1966(later transferred to Altmanella Fend, 2009) has prosoporous atria in VIII; however, it differs from K. hexatheca in most other respects: it is a small worm, without a proboscis, and spermathecae are present only in the postatrial (not in the atrial) segment.The hexathecate condition, with three spermathecal segments beginning with the atrial segment, is only known in Kincaidiana and in Cookidrilus Rodriguez & Giani, 1987.In the latter genus, there is no proboscis, the atria and spermathecae begin in X, instead of IX, and the male ducts are paired and semiprosoporous.Altman, 1936 Figs 1-3

Material examined
Lectotype UNITED STATES OF AMERICA: a sagittally sectioned worm on 4 slides, Series II, from the Altman collection, Washington, Pacifi c County, Loomis Lake, 11 Sep. 1929 (UWBM).

Description of new material
Size variable (Table 2); specimens from swampy habitats usually larger than those from gravel-bed streams; largest specimens (length > 100 mm, diameter > 2 mm, segments > 200, Fig. 1B) from muddy seeps in Oregon.No obvious latitudinal difference in size (see length, width and segment numbers in Table 2).Largest specimens considerably larger than material collected by Altman (1936) (diameter 0.75-1.25 mm) from two sites in Washington and one in Oregon.Proboscis elongate, appearing ringed externally, but lacking internal septa (Figs 1A-B, 2K).
Chaetae in anterior segments (II to VI, VII or VIII) almost always modifi ed, with distal ends oriented anteriad.Modifi ed anterior chaetae may appear simply bifi d in lateral view, with short upper tooth (Figs 1C, 2C), but structure usually more complex: lower tooth broad and fl attened or concave, and thin dorsal keel may extend beyond upper tooth (Figs 1F-G, 2A-B, E-G).Keel visible as translucent outer edge in lateral view, or narrow point in frontal view (Fig. 2F); usually most prominent on replacement chaetae, but absent on many chaetae, possibly due to wear; keel often broken or folded over (Figs 1G,2G) in mounted specimens, with chaeta appearing trifi d.This structure occurs throughout the geographic range of the species, but not always visible in specimens from some populations.Chaetae in atrial and postatrial segments usually simple-pointed, moderately sigmoid and oriented posteriad; tips slightly keeled in posterior segments on some specimens (Fig. 1M).
Chaetae in segment II always smaller than those in next several segments; ventrals in II usually simplepointed.Chaetae from III to about VII or VIII usually thicker than (but similar in length, see Table 2) to those in following segments, with more distal nodulus; distinctly longer in some individuals, and up to 50% longer in Big Creek (west-central Oregon) worms.Within a bundle, inner chaeta typically longer than outer, with more proximal nodulus (Fig. 1C, F).Greatest modifi cation in size, position of nodulus, and development of teeth in anterior chaetae in specimens from Big Creek (Figs 1F-G, 2E-G).
Pharynx with high columnar cells from II to IV, with dorsal and lateral epithelium higher than ventral.Intestine begins in 6/7.Pharyngeal glands in V or VI to VII or VIII, on each side produced into 3 irregular, anteriorly directed lobes (Fig. 2J) joining at the base (posterior septum).Contrary to the description by Altman (1936), they were never observed in II or III, and are about equally distributed dorsally European Journal of Taxonomy 361: 1-46 (2017) and ventrally.Nephridia begin on 11/12, with narrow, dorsally-directed postseptale, as described by Fend (2009).Circular muscle layer of body wall forms distinct bands in anterior segments (Fig. 2L).
Lateral trunks of dorsal blood vessel join to form ventral vessel in III or IV, anterior to location in Altman's description (V).Unbranched, but highly convoluted commissural vessels in anterior segments to about XX.One or two pairs of lateral vessels in segments posterior to about XX; morphology of these vessels variable in middle segments; most commonly, the anterior is larger, branched and covered in chloragogen cells; alternatively, the posterior pair may be branched, and the anterior pair simple, as stated by Altman (1936).Posterior segments may have two pairs of branched vessels.
Male pores always paired, median and posterior to ventral chaetae on IX (Fig. 1A-B); conical porophores developed (Fig. 2N) or not (Fig. 3B-C).Spermathecae paired in IX, X, and XI; spermathecal pores slightly displaced towards ventral midline; pores in IX slightly anterior to chaetae, those in X-XI level with chaetae (Fig. 1A-B).Atria generally more elongate-tubular than in the illustration by Altman (1936: fi g. 59) (Fig. 3).Atrial ampulla and ectal duct weakly differentiated, ampulla distinguished only by slightly greater diameter and presence of prostates (Fig. 2O).Atrium length, including length relative to body width, shows overlap among regions (Table 2).Atria usually entirely in IX, but extend into X in four specimens from sites throughout the species distribution (Fig. 3C).Male funnels may be displaced back within sperm sacs as far as XI (Fig. 3A, C) or even XII.

Remarks
The type series was not clearly designated by Altman (1936), and material used in the original description appears to have been collected from 3 sites in southwestern Washington and northwestern Oregon.The sagittally sectioned specimen here designated as the lectotype (Fig. 2A, K-O) shows most of the diagnostic characters: annulated proboscis, bifi d/keeled anterior chaetae, spermathecae paired in atrial and 2 postatrial segments, conical male porophores, short-tubular atria with prosopore male ducts paired in IX.Other worms from the type series (mostly from undetermined localities) also show these characters (see fi g. 2D-E, G in Fend 2009).
In addition to examining 18 apparent syntypes, Fend (2009) verifi ed diagnostic characters in specimens from additional sites in Washington, Oregon, and California, but did not discuss regional or population differences.Here we examine variation in morphological characters in specimens from throughout the known geographic distribution of the species and molecular differences between specimens from two sites in the southern part of the range, near the type locality of Kincaidiana smithi sp.nov.(see below).
The bifi d, anteriorly-directed chaetae in preclitellar segments are one of the most distinctive characters for K. hexatheca.Despite some population differences in size of these chaetae, the general pattern was consistent in most populations.However, the occurrence of worms from a few sites in southwestern Oregon, having typical K. hexatheca reproductive organs, but with simple-pointed or only slightly bifi d anterior chaetae, cautions against reliance on this character alone for identifying immature specimens.All chaetae (including replacements) were simple-pointed in a partially-mature specimen from Cow Creek (Umpqua River drainage), and immature worms from the same site were similar; chaetae in the fi rst few anterior segments were not enlarged, although they were anteriorly-directed.This condition was variable in mature and immature specimens from two sites in the Rogue River drainage: a spring near Mule Creek (Fig. 1J-K) and the Illinois River (Fig. 1L); in one such specimen the replacement chaetae are bifi d (Fig. 1L), but in other individuals all chaetae (including replacements) were simplepointed.This suggests that simple-pointed chaetae do not simply refl ect wear in worms from gravel-bed streams.
There was no obvious latitudinal difference in size and exent of modifi cation of anterior chaetae (Table 2).The maximum chaeta length in anterior segments of several populations was considerably larger than the 0.218 mm reported by Altman (1936).
Morphology of reproductive organs varied within populations (Fig. 3), possibly masking regional differences.Our observations differ from prior descriptions in minor details.Cook (1971) stated that spermathecal pores are behind the ventral chaetae in IX, but Altman (1936) placed them "between, and just ventral" (median?) to the ventral chaetae.All of the new material has spermathecal pores distinctly displaced towards the ventral midline, and the fi rst pair is clearly anterior to the ventral chaetae (Fig. 1A-B).Atrium length varied by about a factor of 2 within each region, although this was less when normalized by body diameter (Table 2).The limited data suggest that atria of specimens in the California population were larger relative to body size.Spermatheca size varied similarly due to the elongate, irregular ampullae; however, the duct length was less variable.
The species appears to be endemic to the Pacifi c northwestern USA and British Columbia (Kathman & Brinkhurst 1998); reported records from other regions cannot be verifi ed, as material was mostly unavailable for study.Some confusion may be based on ambiguous somatic characters regarded as distinctive in published keys (e.g., Kathman & Brinkhurst 1998).In particular, wrinkling due to fi xation of other proboscis-bearing species may be interpreted as a "pseudo-segmented" proboscis, and minutely bifi d chaetae (possibly a result of wear) on some specimens of Rhynchelmis may also cause confusion.For example, Spencer & Denton (2003) tentatively attributed immature specimens from FEND S.V. et al., Nearctic Kincaidiana and Guestphalinus Utah to K. hexatheca, but recent examination by one of the authors (S.Fend) of some of this material deposited in the Bean Life Science Museum (Brigham Young University, Provo, Utah) suggested that they were more likely to be a species of Rhynchelmis.Therefore, morphology-based identifi cation of immature specimens of K. hexatheca should ideally be based not only on the proboscis and presence/ absence of modifi ed anterior chaetae, but also on other morphological characters.For example, two pairs of lateral blood vessels in segments X-XX of the Nearctic Rhynchelmis species (if present) are short, usually branched, and do not join the ventral vessel (Fend & Brinkhurst 2000).

Molecular data
COI and 16S sequences correspond to topotypic voucher CASIZ 220929 (details in Table 1).

Description
Medium-sized to large worms (Table 2); prostomium short, length about ½ width; fi liform proboscis 1-2 mm long, 0.1-0.16mm diameter, not widened at base, externally ringed with multiple shallow constrictions (Fig. 4A).Body segmentation externally distinct in anterior segments, weak in clitellum and posteriorly; secondary annulation may appear as a narrow posterior ring in V-VII or VIII.Chaetae two per bundle; those in II to (VII)VIII (IX) directed anteriad, others directed posteriad.Chaetae in II or III to VI or VIII appear bifi d with large, fl at ventral tooth and thinner dorsal tooth (Fig. 5D-E some chaetae with a thin dorsal keel above the upper tooth (Fig. 4B).Modifi ed anterior chaetae strongly arcuate distal to the nodulus; shortest in II, gradually increasing in size from III to VI, maximum anterior chaeta length 270-470 μm.Within each pair, median (inner) chaeta has nodulus 0.3-0.4 the distance from the tip; lateral (outer) chaeta may be slightly thicker, with more distal nodulus, 0.25-0.3distance from tip.Chaetae in IV-VII may be longer and thicker (width 15-22 μm) than more posterior chaetae (width 11-15 μm).Posterior to VI-VIII, chaetae usually simple-pointed (Fig. 4B), moderately sigmoid, and directed posteriad; dorsals about the same length as ventrals.Within each posterior pair, lateral chaeta has slightly more distal nodulus.Chaetae in segments posterior to about LXXX may be strongly arcuate; dorsal pairs may appear slightly bifi d or with an upper keel (Fig. 5F).
Epidermis in anterior segments 25-32 μm thick; in clitellum 50-70 μm, thinner in posterior segments.Clitellum from about mid-VII to mid-XIII.In anterior segments, circular muscle layer of body wall arranged in a series of transverse bands with gaps between (Fig. 5B); this layer is 25-50 μm thick in pre-clitellar segments, gradually narrowing posteriorly down to 5-7 μm and appearing homogeneous.Longitudinal muscle layer of body wall 70-100 μm thick in anterior and middle segments.Pharynx from I-III or IV, with dorsal and lateral wall moderately thickened; transition to esophagus indistinct.Pharyngeal glands V-VII, with dorsolateral, median and ventrolateral lobes joining at posterior septa; lobes are joined between segments by thin extensions.No abrupt division between esophagus and intestine.Chloragogen cells cover gut beginning in VII.Brain in the peristomium, not deeply lobed.Dorsal blood vessel separate from gut to about VIII, then closely appressed posteriorly.One pair of commissural blood vessels join dorsal blood vessel near posterior septum between II and about XX; these vessels lack a dense chloragogen layer; in pre-clitellar segments they are long and sinuous; those originating in II-VI join the ventral vessel(s) in the next segment; posteriorly, they are shorter, and join both dorsal and ventral vessels in the same segment.A pair of lateral, blind blood vessels, covered with chloragogen cells, joins the dorsal vessel in the anterior part of segments beginning in about XVI; at fi rst, these are short and unbranched, but by XXV they reach the ventral part of the body, and have up to 10 long branches; by XL they may have more than 20 branches and fi ll much of the coelom.A second pair of blind, branched lateral blood vessels is located in the posterior part of each segment, posterior to about segment L. In more posterior segments (by about C), both pairs of lateral vessels have many short dorsal branches.
Nephridia usually paired on 11/12; occurring irregularly in posterior segments, absent from many segments.A small anteseptal funnel is followed by narrow, dorsally-directed, granular postseptal mass (Fig. 5C).Efferent duct forms a closely-appressed loop, extending to dorsal part of body cavity, forming a convoluted mass; duct ends in a narrow vesicle in front of ventral chaetae in the originating segment; nephridiopore inconspicuous.Male pore single, median and posterior to ventral chaetae on IX; ectal tip of the atrial duct protrudes as a short penis.When everted, penis is subtended by a conical, tiered porophore (Fig. 5M-N), the entire structure up to about 200 μm high by 250-300 μm wide at base; porophore circular, usually consisting of two concentric epidermal folds.Spermathecae paired in IX, X, and XI; spermathecal pores on or very slightly median to ventral chaetal lines, all slightly anterior to respective chaetae (Figs 4A, 5A).One pair of testes in IX, reaching to mid-segment; one pair of ovaries in X, extending to back of segment or into XI; female funnels intersegmental in 10/11, up to 280 μm high.Sperm sacs extend posteriad to XVII-XXIV; no anterior sperm sacs; egg sacs with large eggs extend 1-2 segments behind sperm sacs.

Remarks
Both morphology and molecular results (see below) support K. smithi sp.nov.as a distinct species, closely related to K. hexatheca.Despite the morphological variability of K. hexatheca throughout its broad geographic range, K. smithi sp.nov. is distinctive in that all specimens from the type locality and a nearby site have a single, median male pore and atrium joined by both of the posterior vasa deferentia.The single, median atrium of K. smithi sp.nov. is unusual within the family, but this character appears in both species of Tatriella Hrabě, 1939, as well as in some species of Eclipidrilus Eisen, 1881 (see Fend & Lenat 2012).As in K. smithi sp.nov., this arrangement does not usually represent a simple loss of one of the male ducts from the normal paired condition.In Eclipidrilus pacifi cus Fend, 2005, for example, all four vasa deferentia join the median atrium (Fend 2005: fi g. 9B).Aside from rare and presumably teratological variation within populations, only Eclipidrilus ithys Brinkhurst, 1998 was described as having either one or two atria (Brinkhurst 1998); but when only one is present, the entire male duct is missing from one side.
Compared with most specimens of K. hexatheca, the atrium in K. smithi sp.nov. is more elongate, usually entering the post-atrial segment; nevertheless, this occasionally occurs within the range of variation in populations of K. hexatheca (Table 2, Fig. 3C).The spermathecal pores are clearly displaced from the ventral chaetae line towards the mid-body line in K. hexatheca, while in K. smithi sp.nov. the pores are on the chaetal line, anterior to the chaetal bundles (Fig. 1A-B vs 4A).The morphology of the spermathecae is similar to that of K. hexatheca; however, they are typically more narrow-elongate, with faint distinction between ampullar and duct portions.
Other characters of K. smithi sp.nov.conform closely to those of its congener, K. hexatheca.Both have a large, rather cylindrical body, with a ringed proboscis.The chaetal morphology resembles that of typical K. hexatheca, and the distribution and orientation of modifi ed anterior chaetae are also similar.Less conspicuous, but nevertheless unusual characters are also shared with K. hexatheca (see Fend 2009): the circular musculature of the body wall, the narrow, dorsally-directed nephridia, and the muscle layer extending along the ectal end of the vasa deferentia.

Habitat
The Smith River site is a large (average discharge >100 m 3 /s), free-fl owing stream with riffl e-pool structure and gravel-boulder substrate.Specifi c conductance was 90 μS cm -1 in April 2014 (62-150 μS cm -1 in 1978-1981, NWIS 2016a)).Kincaidiana smithi sp.nov.was only found in a backwater area with some silt deposition.The other collection site was a small roadcut seep, with slow current and some rooted aquatic vegetation.Guts were fi lled with undetermined organic matter and very fi ne mineral particles, with diatoms.Michaelsen, 1933 Type species Dorydrilus (Guestphalinus) wiardi Michaelsen, 1933.

Included species
Guestphalinus wiardi (Michaelsen, 1933) Guestphalinus exilis Fend & Rodriguez sp.nov.Guestphalinus elephantinus Fend & Rodriguez sp.nov.Michaelsen 1933;Cook 1971) Medium-sized to large worms with a fi liform proboscis.Chaetae two per bundle.Paired testes in VIII and IX, one pair of ovaries in X.Male pores paired in IX, near 9/10.Spermathecal pores paired in IX, anterior to the male pores.Petiolate copulatory glands (= Pubertätsdrüsen in Michaelsen 1933) associated with male and/or spermathecal pores.Male duct semiprosoporous.Penes absent.Atria elongate, cylindrical to club-shaped, ental part loosely covered with multicellular, pyriform prostate glands.Anterior vasa deferentia form a loop in the pre-atrial segment before entering the atrial segment.Paired spermathecae in the atrial segment, anterior to the atria.

Distribution
Europe and northwestern USA (present study).Guestphalinus wiardi is rarely reported, but is known from subterranean or spring habitats in Germany (Michaelsen 1933;Griepenburg 1941), Slovenia (Hrabě 1973) and Italy (new material used for the present description, see below), with other records from mountain streams in Crimea (Dembitsky 1987).

Remarks
Among the lumbriculids with a fi liform proboscis, Guestphalinus is distinguished from the semiprosoporous species of Rhynchelmis, Eclipidrilus (Premnodrilus) Smith, 1900 andEremidrilus Fend &Rodriguez, 2003 by the location of the male pores in IX (instead of X), the spermathecae in the atrial segment, the anterior vasa deferentia entering the pre-atrial segment, and the morphology of the elongate spermathecae.In addition to being prosoporous, the two Kincaidiana species are easily distinguished from Guestphalinus by the presence of spermathecae also in the fi rst two postatrial segments, and by a different type of modifi ed chaetae in several pre-clitellar segments.Uktena is distinguished from other proboscis-bearing lumbriculids by having atria and spermathecae in VIII (rather than IX), in addition to characters unique within the family: spermatophores, a spermathecal copulatory organ, and multiple genital chaetae (Fend et al. 2015).

Description of new material and remarks
Hrabě (1973) described a mature, unmated specimen from Slovenia, with sperm on the male funnels.This specimen from Hrabě's collection is reexamined here, but only the anterior body section was available.The fi gures in Hrabě (1973: fi gs 6-7) correspond to photographs 6B and 6C, respectively, in the present paper (note that labels for spermathecal and male pores should be reversed in Hrabě: fi g. 6).The Italian specimen is mature, with clitellum from VIII-XIII and sperm in the spermathecae; the anterior 100 segments are represented.Descriptions by Michaelsen (1933) and Hrabě were quite detailed, and were reviewed by Dembitsky (1987); nevertheless, we can add the following remarks based on both specimens.
A pair of lateral blood vessels is visible in posterior segments of the Italian specimen, and some of these have a few blind branches (Fig. 6I).Chaetae in anterior segments are simple-pointed, but tips of most chaetae in posterior segments bear a distinct dorsal groove, which may appear as a small dorsal tooth in lateral view (Fig. 6F).
The anterior vasa deferentia form a loop in the pre-atrial segment (VIII) before penetrating 8/9 and entering IX to join the atrium (Fig. 6A, E, G) in both of our specimens; this unique character was described and illustrated by Michaelsen (1933), but not mentioned in later descriptions.Posterior vasa deferentia do not penetrate septum 9/10.Michaelsen (1933) described and illustrated a greatly expanded posterior sperm funnel extending well into the posterior sperm sacs.Both posterior and anterior male funnels are small in the Slovenian worm (Fig. 6A); they are much larger in the Italian specimen, and the posterior extends back into X (Fig. 6G).Vasa deferentia are very thick (to 50 μm) in the Italian worm, joining the atrium subapically; they join the atrium before the apex in the Slovenian worm, running a short distance under the muscle layer to enter the lumen apically, as described by Dembitsky (1987) for Crimean specimens.Compared with other descriptions, the atrial ampulla appears rather short and ovate in the Slovenian and Italian worms, although the total atrium length (320 and 335 μm, respectively) and width are similar to those of the Crimean specimens (Dembitsky 1987).Male pores are on a rounded protrusion (referred to as a porophore by Michaelsen 1933), closely behind a groove containing the spermathecal and copulatory gland pores in the Slovenian worm (Fig. 6A-B).Although this structure was also illustrated by Dembitsky (1987: fi g.III) it was not seen in our Italian specimen (Fig. 6G).
Prostate glands are large (to about 100 μm high), petiolate clusters of cells, more similar to fi g. 2 in Michaelsen (1933) than to fi g.III in Dembitsky (1987).
The large copulatory glands (Fig. 6C, H) are quite similar in structure to the copulatory glands described below in the new Nearctic species, although a distinct muscle layer was not seen at the duct.
European Journal of Taxonomy 361: 1-46 (2017) The illustration in Hrabě (1973: fi g. 7) is similar to ours.The slight difference in placement (adjacent to the spermathecae in both of our specimens), compared with Michaelsen's material (at the ventral chaetae), suggests that the position of these glands may be variable, as in Guestphalinus exilis Fend & Rodriguez sp.nov.(see below).Their occurrence may also vary with stage of reproductive development, as Dembitsky (1967) was unable to fi nd these glands in the Crimean specimens, attributing their absence to possible resorption.
The spermatheca is not clearly differentiated into duct vs ampullar sections, although there is a sphincterlike constriction, with thickened circular muscles near the pore of the Slovenian worm (Fig. 6D).
The female funnel is clearly visible only in the Italian worm (Fig. 6G), where it appears to penetrate the posterior septum 10/11 (the plesiopore condition), as in the illustration in Michaelsen (1933).
Guestphalinus was the only lumbriculid genus considered by Brinkhurst (1989: character 16) to have plesiopore female ducts, in common with Haplotaxis, one of the outgroup taxa in that analysis.However, this character has not been observed in G. elephantinus sp.nov.nor in G. exilis sp.nov., where female pores appear to be intersegmental (see below, Fig. 12Q).Plesiopore female ducts appear to be unusual in the Lumbriculidae (Brinkhurst 1989;Fend & Ohtaka 2004), but they may simply have escaped notice in other species descriptions.The exact position of the septum may be diffi cult to defi ne, as muscle fi bers from septa may join the body wall on either side of the female funnel.

Description
Specimens from the type locality: body measurements in Table 3. Prostomium rounded to nearly conical; fi liform proboscis 0.9 to 2.1 mm long, diameter at midpoint 0.1-0.15mm, appearing ringed with multiple shallow constrictions (Figs 7A-C, 9A).Body segmentation not strong in external view; secondary annulation a narrow anterior ring in IV-IX, weak in post-clitellar segments.Clitellum from VIII to XII or mid-XIII, absent ventrally in IX in the area surrounding male and spermathecal pores (Figs 7B, 9A).
Chaetae paired, in 4 bundles in each segment, beginning in II.Chaetal measurements given in Table 3. Chaetae in II bluntly simple-pointed to shallowly notched, directed anteriorly ( Figs 7E-G, 9B-C, E); these chaetae slightly sigmoid, with distal nodulus; within each bundle, lateral chaeta slightly longer than the median, with more distal nodulus.Posterior to II, chaetae sigmoid, mostly simple pointed, with nodulus about ⅓ of distance from tip; perpendicular to body axis or posteriorly directed; chaetae in III slightly shorter, but those in more posterior segments similar in length to those in II.Chaetae in posterior segments may have a slight dorsal keel (Fig. 9D).Ventral chaetae absent in IX in mature and post-reproductive worms.
Epidermis in anterior segments 12-24 μm thick, in clitellum 30-50 μm, posteriorly 5-10 μm.In preclitellar segments, circular muscle layer of body wall arranged in a series of transverse bands (cf.Fig. 12K), 10-15 μm thick; posteriorly a simple layer about 5 μm thick.Longitudinal muscle layer 50-60 μm thick.Brain in the peristomium, lateral lobes rounded.Pharynx begins dorsally and laterally in II, ventrally in III, extending through IV.Pharyngeal glands in IV to VI or VII; on each side, three lobes (dorsal, lateral and median) (Fig. 9G) broadly connected at posterior septum of each segment and extending anteriorly, joining corresponding lobe in previous segment by a thin extension.No abrupt division between esophagus and intestine.Chloragogen cells cover the gut usually beginning in VII; in the most posterior segments many free eleocytes present in the coelomic cavity (Fig. 9H).
First nephridia usually on 6/7, absent in VIII-XI, usually paired on 11/12, occuring irregularly in posterior segments.Each nephridium with small anteseptal funnel; granular postseptal expansion elongate to ovate (length 130-250 μm, diameter 40-80 μm), directed posteriad or somewhat dorsad (Fig. 9I); convoluted efferent duct may pass through one or more adjacent (anterior or posterior) segments, ventral or ventrolateral to the gut, usually near the ventral blood vessel, terminating in a short ectal branch to a simple nephridiopore anterior to the ventral chaetae; indistinct vesicle at the pore in some specimens.Dorsal blood vessel passes under brain: two forks pass around the pharynx and join in IV, forming the ventral vessel.Dorsal vessel free anteriorly, closely appressed to top of gut posterior to VI or VII.Ventral vessel separate from gut, but 2-3 short vessels join it to the perivisceral sinus in each segment posterior to about VII.One pair of commissural vessels in anterior segments; those in II-VI (or VII) long and sinuous, extending through most of originating segment; those in VIII to XII (or XIV) restricted to posterior part of the segment, but a posterior loop from those in IX and X may enter sperm and egg sacs.No lateral blood vessels observed behind about segment XIV.Perivisceral sinus begins in about VII.
Genital fi eld covers ventral side of IX, fl attened or slightly concave in preserved, mature worms.Male and spermathecal pores paired in IX.Male pores near posterior intersegmental groove (Fig. 7A-B) and inside lines of ventral chaetae; spermathecal pores in line with, and in front of male pores, about level with dorsal chaetal bundles (ventral chaetae absent in IX).Female pores paired, on chaetal line at intersegmental groove 10/11.Secretory openings of copulatory glands in IX, small and circular areas, lateral to genital pores, and either in front of or behind them.Paired testes on anterior septa in VIII and IX may be large, extending through anterior half of segment; ovaries in X, reaching to posterior septum, or into XI.Sperm sacs extending back to XIV-XVII in mature worms, egg sacs as far as XV-XVII.
Spermathecae to over 2000 μm long in mated worms, nearly tubular, with two, weakly differentiated sections, both containing sperm.Ectal, duct-like section gradually expanding to form ental ampulla; ampulla may be folded within IX (both in unmated or mated specimens) or extending into X-XII.Ectally, spermatheca passes through a short (12 μm), narrow constriction within ring of muscle fi bers (Figs 8A-B, 9J) and terminates in a shallow epidermal infolding, about 70-110 μm deep.Duct-like section (80-100 μm diameter) with irregular, columnar epithelium (up to 40-45 μm high); dense sperm may be lined up along epithelium (Fig. 9K); very thin outer muscle layer about 2 μm thick.Ampullalike section nearly tubular, diameter about 100 μm for much of its length, but may be entally expanded to over 200 μm (Figs 8A, 9L), with thinner epithelium and wider lumen containing sparse spermatozoa.
Typically, 3-6 stalked copulatory glands are associated with genital pores in mature and nearly-mature worms (Fig. 8A-C); they are absent at early stages of reproductive development.Glands are a pyriform cluster of granular, petiolate cells 160-300 μm long (Fig. 9M-N), sometimes appearing as a group of smaller clusters.Conjoined cell extensions of the gland are constricted by a ring of circular muscle fi bers and then surrounded by thin epithelium before opening in a round secretory surface (to 50 μm diameter) on the body wall.
Anterior and posterior male funnels about equal in size, to 240 μm high; anterior pair rather fl at on septum 8/9; posterior pair on 9/10, but usually directed back into X; both pairs functional, with sperm when fully mature.Anterior vas deferens extends into VIII (Figs 8A-C, 9O), forming a convoluted mass, then penetrates 8/9, running along ventral body wall to near the male pore, then follows atrium to near the ental end, joining the atrium apically.Length of anterior vasa deferentia to 2600 μm, width 36-50 μm.Posterior vas deferens forms a compact, convoluted mass in posterior IX, then follows atrium within the sperm sac, joining it at or near the apical end.Length of posterior vasa over 2000 μm, width 38-46 μm.Vasa deferentia histologically similar throughout, with ciliated, non-glandular epithelium.
Atria of mature worms usually extend back into X or XI; in nearly-mature worms they may be entirely in IX.Atria petiolate in mature worms (Fig. 8A-B); a short ectal duct (150 μm long) has thick, columnar epithelium and a thin (to 2 μm) muscle coat; no distinct penis, although duct may be somewhat expanded at male pore, with thickened epithelium (Figs 8B , 9Q).An abrupt transition from the atrial duct to the tubular or sacciform atrial ampulla.Ampulla length to 1060-1105 μm, width 130-150 μm, thin-walled (5-8 μm) with cuboidal epithelium and wide lumen (Figs 8A, 9P); clusters of prostatic cells, 70-140 μm long, sparsely cover the atrial ampulla.In nearly-mature, unmated worms, atria tubular (Fig. 8C); ampulla and duct not greatly differentiated; prostates small but appear more densely packed than in mated worms.
Eel River, Stony Creek and Garcia River, northern California: The few, partially-mature specimens from these localities are tentatively assigned to Guestphalinus exilis sp.nov.based on the ringed proboscis, the chaetae, and gonads in VIII, IX and X.They differ from the type diagnosis in that chaetae in II are smaller than other anterior chaetae, and simple-pointed (but oriented forward, as in the type locality population).Ventral chaetae in posterior segments distinctly keeled and larger than corresponding dorsals (Fig. 7H-I).As in typical G. exilis sp.nov., specimens from Eel River have semiprosoporous male ducts, with vasa deferentia joining atrium apically and the anterior vas deferens penetrating 8/9 (Fig. 8D).Spermathecal and male pores are behind ventral chaetae of IX and 5 large copulatory glands surround the genital pores.One specimen has an additional, developing spermatheca on one side, in X.

Remarks
Guestphalinus exilis sp.nov.from western North America resembles the Palearctic G. wiardi in characters considered diagnostic for the genus (see above).Compared with G. wiardi, G. exilis sp.nov.has modifi ed chaetae in II, a much larger, sacciform atrium, and more copulatory glands; the male pore was never strongly protruding (Fig. 6B vs Fig. 8A-B).The reproductive organs of G. exilis sp.nov.are morphologically similar to those of G. elephantinus sp.nov.(see below); both lack a distinct male porophore or penis, although the atrial duct may be somewhat expanded at the male pore, with thickened epithelium, suggesting that lining cells may be protrusible (Figs 9Q,12M,O) as "type-2 penes" (temporary structures formed by extruded lining cells of the male duct, see Rodriguez & Giani 1994: fi g. 17D-F).
Molecular results strongly suggest more than one Nearctic species (see below).Both new Guestphalinus species present distinctive chaetal morphotypes, and these chaetal characters are therefore used here as main diagnostic characters.Nevertheless, there is some variation among populations assigned to both basic morphotypes.In segment II chaetae of typical (from Squaw Creek) G. exilis sp.nov.are slightly modifi ed relative to those in other segments.In most Squaw Creek specimens the chaetae appear slightly notched, slightly larger than those in III, and a little shorter than those in middle segments (Table 3).In specimens from two other California sites, chaetae in II are simple-pointed and smaller than those in III -and thus similar to most other lumbriculids.These are provisionally assigned to G. exilis sp.nov., based on the limited and only partially-mature available material.Partially developed reproductive organs (Fig. 8D) clearly indicate that the Eel River specimens belong to Guestphalinus as defi ned here.The single specimens from Stony Creek and the Garcia River, although immature, have gonads in VIII-X, distinguishing them from other proboscis-bearing lumbriculid genera in the region (e.g., Kincaidiana, Rhynchelmis and Eremidrilus).

Habitat
The type locality is Squaw Creek, a tributary to the Sacramento River, in northern California; the collection site is a 5 th order riffl e-pool stream with gravel-cobble sediment (U.S.D.A. 1999).The type series was found only at the lower end of a single riffl e, and no additional material has been found at other sites within the stream.Other oligochaetes were collected throughout the stream; dominant species were Mesenchytraeus pedatus Eisen, 1904 and Rhyacodrilus clio Rodriguez & Fend, 2013 (also the type locality for R. clio).The four streams where G. exilis sp.nov.was collected were similar in size and sediment characteristics, and are coldwater streams, supporting salmonid fi shes.The species was usually found in patches of fi ner gravel within cobble-boulder riffl es.Specifi c conductance at the Squaw Creek and Eel River sites (April 2014) was 265 and 250 μS cm -1 , respectively.Earlier measurements at a downstream site on Squaw Creek (Rettig & Bortleson 1983) gave temperatures from 13.5 to 22.5 °C and a specifi c conductance of 109-261 μS cm -1 .Visits to the type locality from July to September did not produce any specimens of G. exilis sp.nov.The sporadic occurrence and patchy distribution suggest that, like G. wiardi, they may also be primarily groundwater or hyporheic worms that only occasionally show up in surface collections.Fend & Rodriguez sp. nov. urn:lsid:zoobank.org

Etymology
Based on the fanciful and homoplasious similarity to an elephant (proboscis and anteriorly-directed "tusks").

Material examined
Holotype UNITED STATES OF AMERICA: a dissected specimen, slide-mounted in Canada balsam, Washington, Jefferson County, Shale Creek at Clearwater Creek Road, 4 Jun. 2003, S. Fend leg. (USNM 1422286).

Molecular data
COI, 28S and 16S sequences are based on specimens from Shale Creek and Clearwater River, Washington (details in Table 1).An additional ITS sequence (GenBank acc.no.GU592364) corresponds to the Clearwater River voucher SMNH105628.A topotypic voucher is included in the type series (see above).Both COI sequences for G. elephantinus sp.nov.are identical.

Description
Specimens from the Clearwater River drainage, Olympic Peninsula, Washington Description of somatic characters is based on mature or partially-mature (with gonads) worms from Clearwater River, Hurst Creek, Shale Creek and Bull Creek.Reproductive organs are described from the only fully-mature specimen (the holotype), from Shale Creek, and supplemented with 9 postreproductive specimens with partially-resorbed organs from nearby sites Body form and segmentation as described for G. exilis sp.nov.(see above), measurements given in Table 3. Length of proboscis 0.9-2.6 mm (Figs 11A, 12A), clitellum from VIII to XII in the mature worm.Chaetae in II much larger and thicker than those in posterior segments (Table 3), bifi d, with a short, thick lower tooth and a larger, rounded and laterally compressed upper tooth (Figs10A-C, 12C), directed anteriorly, with proximal ends protruding into posterior segments (cf.Figs 11C-D, 12B).Dorsal chaetae in II about 30-40% longer than those in ventral bundles (Table 3); within each bundle in II, the lateral (i.e., outer) chaeta is slightly longer, with a more distal nodulus (0.15 from tip, vs 0.24 in the median (inner) chaeta), and usually has a distal thickening that appears as a "secondary nodulus" between nodulus and tip (Fig. 12C-D).Chaetae in III simple-pointed or slightly bifi d, and oriented more perpendicularly to body, but still facing anteriad (Figs 10C, 12B); much shorter than chaetae in II and slightly shorter than those in other segments (Table 3).Chaetae in IV and posterior segments usually oriented posteriorly, simple-pointed, sigmoid (Fig. 12H).From III to about XX, nodulus about 0.3-0.4 from the tip, but the median (inner) chaeta in each bundle is slightly longer.In posterior segments, chaetae of similar length, but slightly thicker, and nodulus 0.25-0.35from the tip; simple-pointed or with a small notch or dorsal keel in tail segments.Ventral chaetae absent in IX in mature or post-mature worms.
Epidermis in anterior segments 15-20 μm thick; in clitellum to 48 μm in the holotype; narrower, densely packed cells up to 30 μm on ventral side of IX (in area surrounding genital pores) in the holotype and in the post-reproductive worms; posteriorly 10-12 μm.Body-wall musculature, brain, pharynx, pharyngeal glands, nephridia, blood vessels and chloragogen as described above for G. exilis sp.nov., except that pharyngeal glands in G. elephantinus sp.nov.extend to VII in all specimens observed.
Male and spermathecal pores paired in IX.Male pores just anterior to the posterior intersegmental groove (Fig. 11A), slightly inside the line of ventral chaetae; spermathecal pores aligned with and in front of male pores, between or slightly posterior to chaetal bundles.Female pores paired, on chaetal line at 10/11; female funnel up to 130 μm high (Fig. 12Q).A variable number of copulatory glands with internal structure as described for G. exilis sp.nov.; with round secretory openings located close to spermathecal and male pores.The mature worm (holotype) has 3 pairs of petiolate copulatory glands in IX, 100-140 μm high, lateral to, in front of, and behind genital pores (Fig. 11E), although not all of these were visible externally (Fig. 11A).Copulatory glands smaller and indistinct or absent in the postreproductive worms (male duct and spermathecae expanded; large but nearly empty sperm and egg sacs extending to XV), but faint external secretory openings visible in one specimen.
Spermathecae up to 1700 μm long in the holotype; ectal ¼ is duct-like, nearly tubular (about 90 μm in diameter); ental ampulla-like portion irregular, up to about 100 μm in diameter, folded within X or extending into XI.Ectal part with irregularly columnar cells up to 35 μm high, epithelium gradually becoming thinner in ental ampulla.Spermathecae end in a short, narrow constriction surrounded by a ring of muscle fi bers (Fig. 11E, cf.Fig. 12L) which opens in a shallow epidermal infolding, about 40 μm deep.Spermathecae of a paratype similar (Fig. 11G), 1200-2600 μm long, with loose sperm; ampulla thin-walled, ental diameter up to 230 μm, extending into X or as far as XII.
Anterior pair of male funnels on 8/9; posterior male funnels on 9/10, directed back into X; male funnels of post-reproductive worms indistinct, without or with small amount of sperm.Anterior vas deferens extends into VIII (Fig. 11E), forming a compact, convoluted mass, then penetrates 8/9, running along ventral body wall to near the male pore, then follows the atrium before joining the atrial wall and entering the lumen near the apex (junction not clear on holotype).Length of anterior vas deferens up to about 1500 μm, width of both anterior and posterior vasa deferentia 30-36 μm in the holotype.Posterior vas deferens forms a compact, convoluted mass in posterior part of IX, then follows the atrium externally before joining it near the ental end.Atria of holotype extend back into X; ectal duct (length 160 μm, width near midpoint 30-40 μm) has a thick, columnar epithelium and thin muscle coat; ectal part of duct expanded to 50 μm, with epithelial cells directed outward.Entally, there is an abrupt transition to the sacciform atrial ampulla; ampulla length up to 550 μm, width up to 130 μm, with thin-walled epithelium and a wide lumen (Fig. 11E).Small clusters of prostatic cells, 20-40 μm high, very sparse.Atria tubular, with ampulla not expanded in a paratype (apparently at a more advanced stage of maturity, after copulation, Fig. 11G); length 400-800 μm, diameter 60-80 μm; prostates very small or absent; atrial duct and male pore as in the holotype.

Specimens from other localities
Satsop River, western Washington: Modifi ed chaetae in both II and III; chaetae in II very long (Table 3); lateral (outer) chaeta in the largest specimen with secondary nodulus, as in topotypic specimens (Fig. 12D).Chaetae in III smaller, but bifi d and larger than chaetae in the next segments, and directed anteriad (Fig. 11D).
Tucannon River, eastern Washington: Typically modifi ed chaetae in II, but without secondary nodulus; small, simple-pointed chaetae in III; ventral chaetae in posterior segments enlarged, longer and thicker than corresponding dorsals or ventrals in anterior segments posterior to III; some posterior chaetae are slightly keeled (Fig. 10E).In the unmated, nearly-mature worm, tubular spermatheca, vasa deferentia, atrium and prostates similar to those of the Peavine Ridge material (cf.Fig. 11F).Copulatory glands large, up to about 150 μm high, anterolateral and posterolateral to the male pores.Peavine Ridge, northern Oregon: Chaetae in II enlarged and modifi ed as above, without secondary nodulus (Figs 10H,11C); chaetae in III simple-pointed, smaller than other anterior chaetae.Posterior segments with ventral chaetae larger than dorsals; both dorsal and ventral chaetae slightly bifi d or keeled (Fig. 12I).In unmated, nearly-mature worms, there is a variable number (2-4) of well-developed, petiolate copulatory glands with distinct external secretory openings, total height up to 155-290 μm (Fig. 11F).Copulatory glands usually present between spermathecal and male pores (but lateral to both), other glands may be present anterior, lateral, or posterior to genital pores.Much smaller glands may insert at male and spermathecal pores.Atria and spermathecae both tubular, without sperm; male and spermathecal pores (Fig. 12L-M) as described above.
Coastal ranges, central California and southern Oregon: Highly modifi ed chaetae in II as in typical specimens, but without secondary nodulus; the two specimens with the largest chaetae are from this region (Table 3, Fig. 10F, I).Chaetae in III also modifi ed, shorter than those in II but similar in general form (bifi d, with rounded, laterally fl attened upper tooth; nodulus distal); longer than other chaetae in anterior segments (Fig. 12B, F-H).Posterior chaetae usually simple-pointed, ventral chaetae keeled in tail segments (posterior to XC) of some specimens; ventrals in posterior segments larger than those in anterior segments, and larger than corresponding dorsals in some specimens.

Remarks
All populations here attributed to G. elephantinus sp.nov.have highly modifi ed bifi d chaetae in II (or II and III), and their morphology differs from chaetae in all other lumbriculids.These anterior chaetae are distinctly enlarged, very weakly sigmoid, and anteriorly directed with a characteristic short, broad proximal tooth and a laterally-fl attened distal tooth.In comparison, typical G. exilis sp.nov.have similar but only slightly modifi ed (blunt-tipped or slightly notched) chaetae in II.Additional interpopulation variation in chaetal morphology suggests the existence of a species complex, but the rarity of mature specimens from most localities makes it diffi cult to compare populations with the usual morphological criteria, which focus on reproductive structures.Although our limited DNA sampling strongly supports species status for G. elephantinus sp.nov.and G. exilis sp.nov.morphotypes (see below), further molecular studies will be necessary to resolve the diversity of this genus in a region that appears to have extensive radiation within other lumbriculid genera (McKey-Fender & Fender 2001;Fend & Rodriguez 2003).
The morphology of modifi ed chaetae in segment II varies among collection sites, but appears consistent within large series of immature worms collected at some of these sites.Increased chaetal size has been associated with high water conductivity in the Naididae (Loden & Harman 1980, for Pristina aequiseta Bourne, 1891, or for Tubifex tubifex var.grandiseta Rodriguez, 1986).This does not appear to be the case for G. elephantinus sp.nov., since populations with very enlarged chaetae inhabit streams with a wide range of conductivity (<100 to >400 μS cm -1 ).The other western Nearctic species, G. exilis sp.nov., with much less-enlarged anterior chaetae, was collected from streams with intermediate conductivity values (ca 200-300 μS cm -1 , see G. exilis sp.nov.habitat notes, above).
The reproductive organs of G. exilis sp.nov.and G. elephantinus sp.nov.appear similar, although the morphology of these structures is diffi cult to defi ne, as it varies with stage of development.Although atrial morphology is commonly used in defi ning lumbriculid species, it has been shown that it can vary considerably over the reproductive period in Stylodrilus mollis Timm, 1998 and Trichodrilus seirei Timm, 1979.The thin-walled, sacciform atrial ampulla in some mated specimens of both Nearctic species of Guestphalinus appears to be an unusual character for the Lumbriculidae.Unmated, mature worms (Fig. 11F) have tubular atria with a thick epithelium, whereas sac-like atria with thin walls lacking a glandular epithelium or an obvious muscle layer were observed in worms at a more advanced stage of maturity (Fig. 11E, H), including post-reproductive specimens with partially-resorbed reproductive organs.

Habitat
All sites except the spring on Peavine Ridge were alluviated streams with gravel to cobble substrate.All appear to have permanent fl ow, except for Guadalupe and Alamitos Creeks, where surface fl ow may disappear during summer months.Worms were typically collected by digging at least 20 cm deep in patches of fi ner gravel.
The Clearwater drainage sites, on the Olympic Peninsula, northwestern Washington, are larger streams in a watershed dominated by commercial forest, but with riparian buffers.These streams support populations of several salmonid species (Harrington 2005).Limited available water quality data for the Clearwater River near Clearwater (NWIS 2016b) indicate low specifi c conductance (measured in 1972-1974: 13-94 μS cm -1 ).Guadalupe, Alamitos and Euchre Creeks are small, coastal drainages ranging from southern Oregon to central California.The Peavine Ridge collection was from an isolated springfed, seasonally inundated pool with fi ne sediment, and the species was found on only one of several visits.Values for specifi c conductance in both the Peavine Ridge spring and in Euchre Creek (measured in April 2014: 95 and 63 μS cm -1 , respectively) were low.However, values tend to be much higher (at European Journal of Taxonomy 361: 1-46 (2017) summer base fl ow, 340-490 μS cm -1 ) in Guadalupe and Alamitos Creeks (unpublished fi eld data, J.L. Carter, US Geological Survey, 3 sampling dates in May, June, September 1997-1998).

Molecular phylogeny and genetic distances
Two species of Kincaidiana, the two Nearctic species of Guestphalinus and Uktena riparia form a clade with high support: BA posterior probability (BA pp) = 0.99, bootstrap value under ML (ML bs) = 87 in the consensus tree based on concatenated sequence data (COI, 16S rRNA and 28S rRNA) (Fig. 13).Uktena riparia is the sister species to the clade formed by Kincaidiana and Guestphalinus species in the ML tree (not shown); however, in the BA tree (Fig. 13), U. riparia is the sister species to the Kincaidiana species (BA pp = 0.88), with Guestphalinus as the sister group to Kincaidiana + Uktena.Resolving the phylogenetic relationships among these 3 genera needs further molecular work; however, their relatedness is well supported by morphological evidence (see Discussion).
Together, K. hexatheca and K. smithi sp.nov.form a strongly supported clade (BA pp = 1.00,ML bs = 100).The two specimens of K. hexatheca group together (despite morphological differences, already mentioned above), and they are separated from K. smithi sp.nov.The uncorrected (p) distance between the two specimens of K. hexatheca is 3% for COI, and 7.9-8.5% between K. hexatheca and K. smithi sp.nov.; these values are concordant with intra-and interspecifi c values for COI found in other lumbriculids (Achurra & Erséus 2013;Achurra et al. 2015).Note that the type locality of K. smithi sp.nov.(on the Smith River) is geographically located between the localities of the two sequenced specimens of K. hexatheca, and the closest of the latter to the Smith River (Cow Creek, about 150 km to the north) corresponds to the lowest genetic distance (7.9%).For Clitellata, Erséus & Gustafsson (2009) proposed distances of 10% or more for congeneric species; distances of about 5% were interpreted as  (Zhou et al. 2010), which could be the case for the Kincaidiana species under study.Guestphalinus exilis sp.nov.and G. elephantinus sp.nov. group together with high support (BA pp = 1.00,ML bs = 97), having an uncorrected (p) distance of 13.2% for COI; this value is high and together with the morphological differences (mainly based on chaetae, see above) supports the recognition of two distinct species.

Discussion
Both morphology and DNA suggest that Kincaidiana, Guestphalinus and Uktena are phylogenetically related.With respect to morphology, the three genera share several distinctive, and probably synapomorphic characters: an elongate-fi liform proboscis, spermathecae in the atrial segment, male ducts and pores in a segment anterior to X, and elongate or tubular atria and spermathecae.
The fi liform proboscis appears superfi cially ringed in preserved specimens of all three genera, and was referred to as "pseudo-segmented" by Cook (1971) for K. hexatheca, as rings are not associated with septa.Other lumbriculid taxa, Rhynchelmis, Eclipidrilus (Premnodrilus) and Eremidrilus, are also characterized by an elongate, although usually shorter proboscis, and surface wrinkling (which may appear as rings) varies with fi xation.
Most other lumbriculid genera have the spermathecae in either pre-or post-atrial segments, and only the three Cookidrilus species have paired spermathecae in the atrial segment.Two species of Dorydrilus (D. michaelseni Piguet, 1913 andD. tetrathecus Hrabě, 1960) also have spermathecae in the atrial segment.Dorydrilus is generally placed in the Dorydrilidae, although sometimes assigned to the Lumbriculidae (e.g., Hrabě 1983).Cookidrilus and Dorydrilus are small worms, without a proboscis, and also differ from Kincaidiana, Guestphalinus or Uktena in most other respects.
The general form of the elongate spermathecae and atria is similar in Kincaidiana, Guestphalinus and Uktena.In most cases, the ectal, duct-like section of the spermatheca widens gradually to form an elongate, sacciform (irregular) ampulla, and in both Kincaidiana and Guestphalinus the spermathecae terminate in a muscular sphincter just before the pore.As in some other lumbriculids having elongate spermathecae (e.g., some Rhynchelmis (Rhynchelmoides) and Pilaridrilus Fend & Lenat, 2007), dense accumulations of sperm are also lined up along an ectal region with more columnar epithelium, and more diffuse sperm is present in the ampulla (Fend & Lenat 2007, 2010).

Chaetae
Apart from the occurrence of modifi ed genital chaetae in a few species (e.g., U. riparia and four species of Pseudorhynchelmis Hrabě, 1982;Martin & Kaygorodova 2008), most lumbriculids have rather uniform, sigmoid chaetae with a nodulus somewhat distal to the midpoint.Simple-pointed chaetae are most common in the family, although bifi d somatic chaetae occur as intra-generic variation in several lumbriculid genera (e.g., Stylodrilus, Rhynchelmis, Lumbriculus) as well as in the monotypic Wsewolodus Semernoy, 2004.Bifi d chaetae in those taxa have a typical lumbriculid form, with a small, thin upper tooth; furthermore, bifi ds occur throughout the worm, or if variable, teeth are usually more developed European Journal of Taxonomy 361: 1-46 (2017) in posterior segments.Chaetal differences among body regions are usually minor, and limited to greater size of ventrals vs dorsals, or a gradual decrease in size along the body axis.Ventral chaetae may be larger than dorsals in some species of several lumbriculid genera, e.g., Stylodrilus mirus (Cekanovskaya, 1956), Lamprodrilus mrazeki Hrabě, 1928, Sylphella puccoon Rodriguez et al., 2014and Trichodrilus diversisetosus Rodriguez, 1986.The strongly hooked dorsal chaetae of the predaceous Phagodrilus McKey-Fender, 1988 are developed only in posterior segments.Other unusual chaetal modifi cations, such as elongate distal ends of dorsal chaetae in Trichodrilus capilliformis Giani &Rodriguez, 1994 andStylodrilus wahkeenensis Rodriguez &Coates, 1996, the elongated ventral chaetae of Lamprodrilus bythius Michaelsen, 1905 and some Rhynchelmis rostrata (Eisen, 1888) (Fend & Brinkhurst 2000), or the partial or complete loss of chaetae in some species of Lamprodrilus (e.g., L. achaetus Isossimov, 1962) could also be interpreted as derived characters related to a particular lifestyle.Some anterior chaetae are distinctly modifi ed in both Kincaidiana and the Nearctic Guestphalinus; they may appear larger and bifi d or even trifi d in lateral view, and where most developed, modifi ed chaetae in both taxa show a fl attened, dorsal keel.The wide range in size or degree of modifi cation among populations in these taxa suggest local speciation and perhaps adaptation to different external conditions (e.g., sediment particle size distribution).In both Kincaidiana and G. elephantinus sp.nov., the forward orientation of the enlarged and modifi ed anterior chaetae suggests a particular mechanical adaptation to locomotion or feeding (including the possibility of predatory behavior).
The inconspicuous dorsal keel at the tip of posterior chaetae in some specimens of Kincaidiana and the Nearctic species of Guestphalinus has not been reported in other lumbriculids, and the grooved tips on posterior chaetae of one specimen of G. wiardi also appear unusual.Nevertheless, similar chaetae with keeled tips have been described in unrelated taxa, e.g., two species in the family Haplotaxidae: Delaya leruthi (Hrabě, 1958) (Delay 1972: fi gs 1-2) and D. navarrensis (Delay, 1973) (Delay 1973: fi g. 1).

Male ducts
Guestphalinus has a semiprosoporous male duct (with 2 male ducts per atrium), in contrast to the prosoporous ducts of the related Kincaidiana and Uktena.Although this has been considered a phylogenetically important character, it is likely that a reduction of the anterior vas deferens has occurred more than once in lumbriculids (see Brinkhurst 1989).
Both of our specimens of G. wiardi and the description by Michaelsen (1933: fi g. 2) indicate that the anterior vasa deferentia form a loop in the pre-atrial segment, as in G. exilis sp.nov.and G. elephantinus sp.nov.This character has not been reported in other Lumbriculidae so far.Hrabě (1984) considered the penetration of the posterior septum by the posterior vas deferens in semiprosoporous lumbriculids to be an atavistic character, associated with the loss of the posterior pair of atria from a double-pair of prosoporous atria; following that logic, this new structure could suggest the loss of an anterior atrium.The male funnels extend several segments back within posterior sperm sacs in Kincaidiana, Uktena, and possibly in some G. wiardi (Michaelsen 1933: fi g. 2; but not confi rmed in our material); this character is also somewhat unusual in the family, even in genera having very elongate atria (e.g., Rhynchelmis and Eclipidrilus).

Copulatory glands
Large, stalked glands (Pubertätsdrüsen in Michaelsen 1933), are associated with the male and/ or spermathecal pores in Guestphalinus.The glands have a similar structure in all species, with cell extensions passing through a narrow muscular constriction and terminating in small, circular secretory openings in the epidermis.These glands are here regarded as probable copulatory glands, as they are well developed in both nearly-mature (unmated) and mature worms, often smaller in post-copulatory worms with well-developed eggs, and disappear in post-reproductive worms.Species of Kincaidiana have well-defi ned penes, the most common condition in Lumbriculidae, but the absence of this effi cient FEND S.V. et al., Nearctic Kincaidiana and Guestphalinus means of sperm transfer in Guestphalinus further suggests that the glands in the ventral region (close to the genital pores) are associated with mating.Large glands of this form also occur in Uktena riparia Fend et al., 2015, where they probably have some function in the transfer of spermatophores, but, instead of opening externally, these open within the complex spermathecal and male bursae.Glands of this form are uncommon in the Lumbriculidae, but similar structures, with external openings in (or just behind) the male segment, have been described in Rhynchelmis brooksi Holmquist, 1976 and some species of Lamprodrilus (e.g., Isossimov 1962: fi gs 14, 28), where they have been termed "accessory copulatory glands" (Cook 1971;Michaelsen 1901).Midventral glands with a similar form have also been described in anterior segments of sexually mature specimens of some species of Rhynchelmis (Rhynchelmoides) (Holmquist 1976;Fend & Brinkhurst 2000, 2010).All of the above glands lack a distinct duct, and the conjoined ends of the gland cells open via a small secretory surface.Members of other oligochaete families (e.g., Naididae, Enchytraeidae, Haplotaxidae) have similar glands opening externally near the genital pores at maturity, in some cases associated with genital chaetae (e.g., Rodriguez & Fend 2013); their sporadic occurrence in different lineages suggests that they are convergent.
Glands associated with reproductive structures in some other lumbriculid genera differ in structure.More elaborate, glandular "Kopulationsdrüsen" (e.g., Michaelsen 1901), or "accessory organs" (Fend & Brinkhurst 2010) occur in some Rhynchemis (Rhynchelmis) species.These organs have an internal lumen, opening through a duct into a simple pore, and have been considered to be rudimentary atria.Michaelsen considered the glands in G. wiardi as possible "Geschlechtsborstendrüsen", similar to glands associated with copulatory chaetae in other microdriles, but he was unable to associate them with the ventral chaetae.Although common in other microdrile families, glands associated with genital chaetae are rare within the Lumbriculidae, and have only been described in detail for some species of Pseudorhynchelmis Hrabě, 1982(Martin & Kaygorodova 2008).

Conclusions
Morphology (proboscis, spermathecae in the atrial segment, and general morphology of reproductive organs) suggests that Kincaidiana, Guestphalinus and Uktena are phylogenetically related.This relationship is confi rmed by the molecular data available at this time: the three genera form a strongly supported clade in the tree topology (Fig. 13), based on a limited set of lumbriculid outgroup taxa.At this point, it should be noted that no molecular phylogeny of the family Lumbriculidae as a whole has been published.Further molecular analyses, including more genera, are clearly a priority for any understanding of relationships within the family.
Populations of both Kincaidiana and Guestphalinus are quite variable across their distributions in western North America.Our DNA results provide good confi rmation that the two most distinctive populations, K. smithi sp.nov.(with a single median atrium) and G. exilis sp.nov.(with only slightly modifi ed anterior chaetae), are closely related to their more widespread congeners, K. hexatheca (with paired atria) and G. elephantinus sp.nov.(with highly modifi ed anterior chaetae), yet differ by interspecifi c COI distances of 8% or more.Still, given the geographic variation in morphological characters in Nearctic populations of Kincaidiana and Guestphalinus, further molecular analyses among and within populations would also be of interest, and could provide evidence of speciation processes in the region.Finally, behavioral observations, including the feeding behavior of Kincaidiana and Guestphalinus, may eventually reveal the function of modifi ed chaetae.

Fig. 4 .
Fig. 4. Kincaidiana smithi Fend & Rodriguez sp.nov., from the type locality, except for C, from seep by South Fork Smith River, CA. A. Complete worm, lateral and ventral views, showing location of reproductive pores.B. Representative chaetae: Roman numerals indicate segment number; dorsal chaetae with tips upward, ventrals with tips down.C. Reproductive organs in IX-XI.D. Reproductive organs in IX-XI, from a sagittal section (middle portion of atrium missing).E.An atrium, joined by both vasa deferentia.F. A spermatheca.

Fig. 5 .
Fig. 5. Kincaidiana smithi Fend & Rodriguez sp.nov., from the type locality.B-C, I-L, N-O from sagittal sections; D-H, M from dissections.A. Ventral view of an entire worm, stained with hematoxylin, showing positions of genital pores.B. Body wall in segment III, showing longitudinal and transverse muscle layers.C. Nephridial funnel and postseptal expansion.D. Chaetae in V dorsal.E. IV ventral.F-G.Chaetae in posterior segments; (F) is detail of tip, showing slight keel.H. Spermatheca.I. Junction of spermathecal "duct" and "ampulla"; sperm are in the ampullar part.J. Ectal end of spermathecal duct, with sperm lined up along epithelium.K. Spermathecal vestibule and ectal end of duct, with muscular sphincter.L. Atrium with prostate glands.M-N.Penis and male porophore.O. Vas deferens joining atrium near middle.Scale bars: A = 1 mm; B-C, G, J-L, O = 100 μm; D-F = 50 μm; H-I, M-N = 200 μm.

Fig. 6 .
Fig. 6.Guestphalinus wiardi (Michaelsen, 1933); A-E a sectioned worm from Slovenia, Hrabě's collection; F-I, a dissected Italian specimen.A. Male duct and spermatheca in IX, reconstructed from sections; pore from a copulatory gland (not shown) opens next to spermathecal pore.B. Atrium and male porophore.C. Copulatory gland.D. Spermathecal pore and duct.E. Anterior male funnel with vas deferens entering pre-atrial segment.F. Posterior ventral chaeta, showing grooved dorsal tooth.G. Reproductive organs in IX-XI, fi gure rotated to face to right.H. Detail of G, showing position of copulatory gland, from median view.I. Branched lateral blood vessel in posterior segment.Scale bars: A, G-H = 200 μm; B, E, I = 100 μm; C-D, F = 50 μm.

Fig. 7 .
Fig. 7. Guestphalinus exilis Fend & Rodriguez sp.nov., external characters.A-C, E-G from Squaw Creek, CA (the type locality); D, H from South Fork Eel River, CA; I from Stony Creek, CA.A-B.Ventral views of two worms, showing positions of genital pores: A is partially-mature, with 3 glands opening around male and spermathecal pores; B is from a reproductively-mature worm, dashed line encloses the non-clitellar genital fi eld with 5 glands opening around spermathecal and male pores.C-D.Anterior end, showing orientation of chaetae.E-G.Selected chaetae from typical specimens; G shows relative position of dorsal chaetae in II and III.H-I.Selected chaetae from partially-mature worms.Chaetae are numbered by segment (Roman numerals), followed by "d" (dorsal) or "v" (ventral).Scale bars: A-B = 2 mm; C-D = 1 mm; E-I = 100 μm.

Fig. 8 .Fig. 9 .
Fig. 8. Guestphalinus exilis Fend & Rodriguez sp.nov., reproductive structures, all oriented with anterior end to left.A-C from Squaw Creek (the type locality), D from the South Fork Eel River A. Segments VIII-XI of a mature worm, showing reproductive organs; anterior vas deferens broken at spermatheca.B. Segments IX-X of a recently-mated worm; spermathecal ampulla not yet developed; atrium expanded.C. Segments IX-X of a partially-mature worm; atrium and spermathecae tubular, but copulatory glands well-developed; ental end of spermatheca missing.D. Segment IX of a partially-mature worm; glands well developed; atrium and spermatheca small, tubular.

EuropeanFig. 10 .
Fig. 10.Guestphalinus elephantinus Fend & Rodriguez sp.nov., various sites, arranged approximately north-south; paired chaetae; d = dorsal bundle, v = ventral.A. From Shale Creek, WA (type locality).B-C.From Clearwater River, WA; C shows II and III in relative positions at top.D. West Fork Satsop River, WA.E. Tucannon River, WA; II dorsal bundle with 1 replacement chaeta.F. Euchre Creek, OR.G. Mule Creek at Rogue River, OR; II and III dorsal are shown in relative positions.H. Spring at Peavine Ridge, OR.I-J.A mature and immature worm from Guadalupe Creek, CA; II and III shown in relative positions; XL ventral and C ventral in I have partially-developed replacements; II ventral chaetae partially-developed in J. Scale bars: 100 μm.
Fig. 11.Guestphalinus elephantinus Fend & Rodriguez sp.nov. A. Ventral view of two worms from Shale Creek, WA (type locality); upper drawing is the holotype, lower is a post-reproductive worm.B. Ventral view of segment IX, showing genital fi eld of a mature worm from Guadalupe Creek, CA; secretory openings of 5 glands are visible, in addition to paired male and spermathecal pores.C. Anterior end, from Peavine Ridge spring, OR, showing position of chaetae in II-IV.D. Anterior end of worm from Middle Fork Satsop River, WA.E. Reproductive organs of a mated worm from Shale Creek (holotype).F. Segment IX of an unmated worm, Peavine Ridge.G. Atrium and spermatheca in IX of a mated, but slightly post-mature worm from Clearwater River, WA; sperm present in spermatheca, but atrium is reduced in size.H. Reproductive organs in segments VIII-XII of a mated, mature worm from Guadalupe Creek, CA.

Fig. 13 .
Fig. 13.BA consensus tree derived from the concatenated sequences of COI, 16S rRNA and 28S rRNA genes.Numbers beside internal branches indicate BA posterior probabilities (≥ 0.90) and ML bootstrap values (≥ 70%).The geographical location for two specimens of K. hexatheca Altman, 1936 is given under the taxon name.