A new species of Speonemadus from Portugal with the revision of the escalerai-group (Coleoptera: Leiodidae)

. Over recent years, intense field work in caves of Portugal has provided new data on the distribution of subterranean Iberian leiodid beetles. Speonemadus algarvensis sp. nov. is described from caves of southern Portugal. The new species is included in the Speonemadus Jeannel, 1922 escalerai group (Cholevinae; Anemadini). All species of the S. escalerai -group are revised and S. breuili (Jeannel, 1922) is res urrected as a valid species. A key to identify the species of the S. escalerai -group is provided and the distinctive characters are illustrated. The distribution of all species of the group is mapped with new data together with biogeographic considerations.


Introduction
The genus Speonemadus Jeannel, 1922 has been the subject of a comprehensive review by Giachino & Vailati (1993), who established Hormosacus Jeannel, 1936 as a synonym of Speonemadus, due to close structural homology between morphological characters of the aedeagus.
We describe a new southwestern Iberian species of Speonemadus, that colonizes caves of southern Portugal, and revise all species of the escalerai-group.We also resurrect Speonemadus breuili (Jeannel, 1922) as a valid species.

Material and methods
Specimens were collected with baited pit fall traps and by active searching in several caves of the Iberian Peninsula (Reboleira 2012).
Male genitalia were extracted and immersed in a 10% KOH solution for six hours and then dehydrated with a series of ethanol transfers (60% to 96%), followed by a xylene immersion for 12 hours and final mounting in Canada balsam.
The general external morphology of the specimens was photographed with a stereo microscope Olympus SZX16, the aedeagus with an Olympus CH; both using an Olympus C5060WZ camera; photos were combined using CombineZP.For scanning electron microscopy, specimens were critical point dried in a Tousimis Autosamdi 815, series A, and mounted on aluminium stubs, coated with platinum/palladium and studied in a JEOL JSM-6335F scanning electron microscope.Images were processed with Adobe Photoshop CS6.
Habitus length was measured from the apex of the labrum to the tip of the elytra.Mean measurements in Table 1 correspond to six specimens of each sex.

Institutional abbreviations
The material is deposited in the following collections:

Results
Class Insecta Linnaeus, 1758 Order Coleoptera Linnaeus, 1758 Family Leiodidae Fleming, 1821 Subfamily Cholevinae Kirby, 1837Tribe Anemadini Hatch, 1928 Genus Speonemadus Jeannel, 1922 Speonemadus escalerai-group; "Gruppo escalerai" sensu Giachino & Vailati 1993: 205.Representatives of this group are characterized by a body length between 3.9 to 5.0 mm and have a slightly transverse pronotum ; the anterior tibia of the males is compressed towards the apical half and has a keel with different shapes on the dorsal edge ; elytra have a rounded apex in males and a notched one in females.The median lobe of the aedeagus is elongated and bottleshaped in dorsal view  and the inner sac lacks a ventral tooth in the apical region of the middle lobe and has two long parallel rows of fine spines and teeth .
All species of the group have similar color patterns, the same antennal shape and development of the membranous wings in both sexes, as well as similar genital segments and aedeagi in males, and similar 7 th and 8 th urosternites and spermatheca of females.
The life-style of this group is mostly subterranean, they are frequently found under stones or in leaf litter in forests and in caves, walking in bat guano or in the subterranean substrate.

Key to the species of the Speonemadus escalerai-group
Note to the key: diversification of the species of the escalerai-group is likely to be recent (see below "Speciation and biogeography").Therefore, morphological differences are not particularly visible and a combination of characters should be used for species recognition.All species and both sexes of the "escalerai" group show a similar coloration, shape of the antenna, development of membranous wings, genital segment in the male aedeagus and 7 th and 8 th uroventrites, as well as spermatheca of the females.
Females have no characters to distinguish between species, which also occurs in other genera of the Leiodidae, e.g., Colon Herbst, 1797.Only females of S. angusticollis can be separated from other species by the shape of the pronotum, being the only one with a bell-shape, as in males.Therefore, in this species group only males provide distinctive characters useful for identification.These characters are identified in all key items and constitute the basis of specific differentiation.2. Protibial keel with sharp vertex and slightly lobed from the margin to the base (Fig. 20); elytra sculpture and pronotum granularity poorly marked; pronotum sexually dimorphic (Figs 14-15) …… …………………………………………………………………………S.escalerai (Uhagón, 1898) -Protibial keel with a slightly sharp vertex and linear margin to the base (Figs 16,18); elytra sculpture and pronotum with well marked granularity; pronotum not sexually dimorphic (Figs 6-7, 10-11) …3 3. Body size generally small (male length around 4.2 mm); protibial keel with sharp apex and basal part of the protibia with a wide keel (Fig. 18); 8 th antennomere around 1.5 × longer than wide and 4 th one length different from 5 th and 7 th antennomeres in both sexes …S.bolivari (Jeannel, 1922) -Body size generally larger (mean male length around 4.8 mm); protibial keel with rounded apex and the basal area of the protibia with a wider keel (Fig. 16); 8 th antennomere more than 1.4 × longer than wide and 4 th antennomere equal or almost equal in length to 5 th and 7 th antennomeres in both sexes …………………………………………………………………S.algarvensis sp.nov.

Diagnosis
A species of Speonemadus with a total length of 4.0-4.9mm, belonging to the escalerai-group, and with typical features of the group; characterised by having the 2 nd , 4 th , 5 th and 7 th antennomeres equal or subequal in length; a slightly transverse and hexagonal pronotum; and a long, raised and elevated protibial keel in males, with the apex moderately sharp.

Male
Some variability is observed in male paratypes (Table 1).Genital segment elongate, 1.5 × longer than wide; composed of a tergum and two pleurites, all with small setae in apical area; sternum reduced to a long, narrow longitudinal piece, sharp apically.Aedeagus length = 1.2 mm (1.3 if parameres are included) (Fig. 21).Median lobe, lanceolate in dorsal view, with elongate and curved apex close to dorsal face; sharp apex with marked shoulders at margins; basal lamina developed, almost two times shorter than middle lobe; ventral blade of tegmen short and poorly defined.Parameres, in dorsal view, sub rectilinear, slightly curved medially on inner side, with apical areas rounded and slightly truncated on inner side; four thin setae at apex, three subequal upper setae and a larger one, close to largest setae, another seta, shorter, more robust, tooth-like and highly sclerotized.Inner sac with two long rows of spinules surrounding two long parallel rows of sclerotized teeth arranged in a zipper-shape (Figs 21,31).

Affinities
The morphological differences that separate Speonemadus algarvensis sp.nov.from all other species of the S. escalerai-group are pointed out in the key for the group.
Morphologically, the most closely related species are Speonemadus bolivari and S. breuili, which are also the closest geographically (Fig. 32).However, S. algarvensis sp.nov.can easily be distinguished from S. bolivari by the different shape of the male protibial keel, by the antennomeres ratio and the shape of the aedeagus, with the median lobe apically more narrow and elongate.Differences can also be observed in the females of S. algarvensis sp.nov., with the median posterior slit of the 7 th uroventrite slightly narrower and the apex of the ventral spine of the 8 th uroventrite less sharpened.The differences regarding S. breuili are much more clear in the shape of the protibial keel.
There is a strong geographic isolation between karst areas colonized by the previous three species, which have the most southern distribution of the group (Fig. 32).
The male specimen collected on 4 Jan.1940, in the cave Igrejinha da Soídos, located in Alte, municipality of Loulé (Jeannel 1941), deposited in the Natural History Museum of Paris (MNHN), has been examined and belongs to the new species S. algarvensis sp.nov., it had previously been identified as S. angusticollis.The remnants of another specimen collected in the cave "Berrocal do Esguincho", also in the Loulé municipality, on 6 Dec. 1983 and cited by Blas (1985Blas ( , 1989)), are also included in this new species.Both aforementioned caves are included in the same karst area as the type localities of S. algarvensis sp.nov., where only this species of Speonemadus has consistently been collected.The specimens reported from the Algarve massif as Speonemadus angusticollis by Jeannel (1941), Blas (1985Blas ( , 1989)), Reboleira et al. (2010aReboleira et al. ( , 2010bReboleira et al. ( , 2011bReboleira et al. ( , 2012a) ) and Reboleira (2012) should now be included in S. algarvensis sp.nov.

Biology and ecology
Despite lacking some of the typical traits of cave-adapted species, i.e., eyes, severe depigmentation and extreme body and appendages elongation, Speonemadus algarvensis sp.nov.was never found at the surface, leading us to classify it as a subterranean species.It has been sampled all year round and it has never been collected at the surface in the areas surrounding the sampled caves.The largest population was found in Vale Telheiro Cave, which matches with the general high biodiversity pattern for other groups of troglobionts in Portugal (Reboleira 2012;Reboleira et al. 2015).Some specimens of S. algarvensis sp.nov.have the ectoparasitic fungus Stichomyces conosomatis Thaxt., 1901 (of the order Laboulbeniales) attached to the cuticle (Santamaria pers.com.) (Fig. 26); this represents the first record of the fungal species for the Portuguese territory.This fungus species has only been previously found in Staphylinidae beetles of the genus Sepedophilus Gistel, 1856 (Haelewaters et al. 2015), that was also found in these caves (Reboleira unpublished).The remarkable discovery of this Laboulbeniales species on a new host of the family Leiodidae is the first case of host shifting following an ecological opportunity (sensu De Kesel & Haelewaters 2014), in the subterranean environment.Also phoretic undetermined mites were observed on some specimens (Fig. 27).

Distribution
Speonemadus algarvensis sp.nov.was collected in three caves of the southernmost province of Portugal, the Algarve (Fig. 32), where it seems to be endemic to the central and eastern part of the Algarve karst massif.The species was not found in the most western part of the Algarve massif, where fieldwork was also conducted.The karst included in this region is also know as "Barrocal" and is the richest area for subterranean-adapted fauna of the country (Reboleira 2012;Reboleira et al. 2011aReboleira et al. , 2013Reboleira et al. , 2015;;Reboleira & Enghoff 2013, 2014).(Kraatz, 1870) Choleva angusticollis Kraatz, 1870: 98.
Pronotum .Slightly transverse with surface slightly punctured , maximum width/length ratio = 1.20-1.32(males) and 1.18-1.25 (females), with maximum width in the middle third and the lateral edges regularly arcuate, converging towards the base; posterior angles obtuse and slightly rounded at apex.Pronotal base sinuate and narrower than elytral base.
Elytra.Elliptical and very elongated, maximum length / width ratio = 2.04-2.19(males) and 1.97-2.11mm (females); apex of each elytron rounded; elytral disk slightly convex and flattened in the middle.Elytral sculpture formed by very strong sutural striae and transverse strias with regular arrangement, sunken, well marked and perpendicular to the suture.

Male
The structure of genital segment corresponds to the typical pattern of the genus, formed by an urotergite with the apex in sharp bow, two uropleurites with apically narrow, sharp, slightly outwardly and short shaped handle ventrite.Aedeagus robust and long, between 1.2 and 1.3 mm (Fig. 22); basal lamina almost 2 × shorter than middle lobe; ventral blade of tegmen short and poorly defined.Median lobe in dorsal view, similar to other species of the group, although it has the triangular apex with less protruding edge and narrower at its distal third part.Parameres robust and wide with a marked hump on the outside of distal and the apical area is beveled with a blunt tip, in this area four setae are inserted, of which three are short and thin, and the fourth is longer with a small tooth, coarse and finely sclerotized.Inner sac showing two longitudinal chains of small sclerotized pieces, surrounded by a longer structure formed by numerous fine spines that are joined at the apex, but not tooth-shaped.Legs long with a peculiar structure of the keel protibial (Fig. 17) which is long, slightly elevated, with a straight area, and fuzzy projections; in addition, the first three protarsomeres are dilated, although first protarsomere is narrower than anterior area of protibia; mesotibia arched and metatibia straight.

Female
The most significant external differences are observed on the antennae, pronotum, elytra, protibia and protarsomeres.Antennae very similar to those observed in males (Table 1); the 2 nd antennomere equal to the 5 th one, and the 8 th almost 1.5 × longer than wide.Pronotum slightly transverse and clearly narrower than in males (Fig. 9), its sides almost straight and divergent on the posterior part; the maximum width is near the base and bell-like in form; posterior angles are almost straight with rounded angles; pronotal base is almost as wide as base of elytra.Elytra are elliptical and elongated and proportionally slightly shorter than in males.Apex of elytra is notched and toothed.Protibiae lacking keel and protarsi slender.7 th and 8 th uroventrites and genital segment typical of the genus (see Giachino & Vailati 1993).

Variability
The most significant variation was observed in the samples from the two caves of Huelva (Gruta de la Maravillas and Cueva del Guerrero), specimens from both caves show intermediate characteristics between S. angusticollis and S. breuili, with the protibial keel more close to the first one and female pronotum most similar to the second species.Systematic position of the specimens collected from these two caves will be solved only by further study of specimens from caves near the Sierra de Aracena.

Biology and ecology
This species is found in a forest environment, in leaf litter or under stones.In arid climates it tends to colonize the subterranean ecosystem, being frequently found in caves, as in Spanish Andalusia and now also in the Portuguese region of Alentejo.In less dry regions it is found to be more associated with the soil ecosystem (Blas 1977).It has been also been reported (Giachino & Vailati 1993) from the Superficial Subterranean Habitat (sensu Giachino & Vailati 2010).

Distribution
Described from Spanish Córdoba (Kraatz 1870), this is an Iberian endemic with a scattered distribution in the central area of the peninsula, being more common in its southern and western parts.It is known from the Spanish provinces of Albacete, Ávila, Badajoz, Cáceres, Ciudad Real, Córdoba, Huelva, Jaén, Madrid and Sevilla and now also from southeastern Portugal (Fig. 32).The present finding in a cave of Alandroal, in the Alentejo province, is the first confirmed record of this species for the Portuguese territory as previous records for Portugal (Jeannel 1941, Blas 1985, 1989, Reboleira 2012, Reboleira et al. 2010a, 2010b, 2011a, 2012a) refer to S. algarvensis sp.nov.(Jeannel, 1922) Anemadus (Speonemadus) breuili Jeannel, 1922: 60.

Redescription
Body.Length 4.0-4.5 (males) and 3.9-4.7 mm (females), body width 1.4-1.5 (males) and 1.5-1.7 mm (females) (Fig. 4).Body color, pubescence and punctuation similar to that observed in S. angusticollis.antEnnaE.Reaching the basal 4 th part of elytra; the antennomeres 1 st and 2 nd are almost equal in length, also equal in length are the 4 th , 5 th and 7 th ones being the 3 rd ones clearly longer than the 2 nd one and the 8 th nearly twice as long as wide (Table 1).
Pronotum.Slightly transverse, maximum width at middle third part; the angles converge, smoothly from the middle area to the base in a straight line; posterior angles obtuse and rounded at vertex; sculpture and granularity well defined (Fig. 12).
Male (Fig. 24) Features of the genital segment and aedeagus are similar to those described for S. angusticollis.

Female
The antennae are similar to those of the male, but the antennomeres are more transverse; 2 nd , 4 th , 5 th and 7 th antennomeres having similar length (Table 1).Pronotum less elongated; the overall shape and the ratio of width / length ratio similar to that described for males (Fig. 13).Elytra elliptical and elongated with apical parts notched and serrated.7 th and 8 th uroventrites, and genitalia typical of the species group.

Remarks
Speonemadus breuili is re-established as a valid species due to the clear morphological characters that differentiate it from S. angusticollis, which was synonymized by Blas (1989).This classification was subsequently followed by several authors (Giachino & Vailati 1993;Perreau 2000;Salgado et al. 2008).The main character used to re-evaluate its specific status is the shape of the male protibial keel, which is shorter and with a more rounded ridge, but also because in S. breuili the 8 th antennomere is less transverse, the elytra are proportionally shorter, and the female pronotum is more hexagonal and clearly transverse.In addition, Speonemadus verneri is established as a synonym of S. breuili and not of S. angusticollis (Blas 1989), due to the absence of relevant differences compared to S. breuili.
Apart from the morphological differences pointed out, there is an obvious geographical isolation.Speonemadus breuili colonizes the southernmost caves in Spanish Andalusia, the provinces of Cadiz and Málaga, away from the distribution area of S. angusticollis.antEnnaE.Long, slender, light brown and extended beyond the basal fourth of elytra; 3 rd antennomere slightly longer than the 2 nd one; 6 th one shorter than 4 th and 5 th ones, and the latter one as long as the 7 th one; 8 th almost 1.5 × longer than wide (Table 1).
Pronotum.Slightly transverse, maximum width / length ratio = 1.23-1.39(males) and 1.28-1.36mm (females); with a maximum width towards the middle; basal impressions are not hardly noticeable; side edges regularly arched forward, almost straight and towards the posterior angles that are obtuse and rounded in the vertex (Fig. 10); pronotal base as wide as elytral base; sculpture has a coarse granularity with obvious microsculpture.

Male
Genital segment very similar to the other species of the escalarai-group and following the model described for Speonemadus algarvensis sp.nov.Aedeagus long, 1.1 mm; basal lamina of median lobe well developed and more than half of the median lobe length; ventral blade of tegmen short, inconsistent and forms the widest part of the aedeagus.In dorsal view, median lobe is bottle-shaped, with converging sides towards the base and narrowed distally; apex tapers and ends in a triangular tip with the sides slightly beaded dorsally (Fig. 23).Parameres robust, only curved forwards; apical parts obliquely truncated on the inner side, where four setae are inserted, three thin, subequal, and a larger one, as well as a thick and well sclerotized small tooth.Inner sac with two longitudinal chains formed by small sclerotized parts and surrounded by two longer chains of fine and sharp spines that come together in the apical region, but do not form a typical sclerotized tooth as in other species.lEgs.Quite long; the protibial keel is elevated in the anterior middle third, showing a single, well marked, projection angle (Fig. 18); first three protarsomeres dilated, being the first almost as wide as the apical tip of the protibia; mesotibia strongly arched and metatibia straight.

Female
Antennae slightly shorter and more robust than in the male, reaching third basal part of elytra; 2 nd antennomere is slightly longer than the 3 rd one and 5 th one longer than 4 th and 6 th ones; the 8 th being clearly longer than wide (Table 1).Pronotum less transverse, but appears longer (Fig. 11).Elytra elliptical and elongated with notched and serrated apical areas.The sexual dimorphism is clear for all species of this group, as well as the structures in uroventrites 7 th and 8 th , and spermatheca model.

Biology and ecology
Subterranean species, found in deep areas of scattered caves, under stones or on calcite mantles.Groups of adults and larvae have been found in small accumulations of organic matter, such as bat guano.

Distribution
Speonemadus bolivari is endemic to several karst caves located in a narrow strip within the province of Málaga, between the Serrania de Ronda, at the western limit, and the karst areas at its border with the province of Granada, such as the Romeral complex, at its eastern limit.The three distribution records for the Granada province are located to the north and east of the massif of the Sierra Nevada (Fig. 32).(Uhagón, 1898) Anemadus escalerae Uhagón, 1898: 118.

Male
Aedeagus and structures of inner sac are similar to those described for S. bolivari, although larger in shape, length 1.4-1.5 mm (measured to the apex of the parameres); in dorsal view very similar to all the species of the group but in close slimmer apical area (Fig. 25).Parameres also similar to S. bolivari, but with more pronounced steeper area of outer margin in third distal part.gEnital sEgmEnt structurE.Similar to that described for Speonemadus algarvensis sp.nov.lEgs.Relatively long and slender; protibia widened in apical area and with a long keel running through the two basal thirds of the margin, showing two sharp protrusions, separated by a slight depression, anterior is sharp and much higher and the second is fuzzy (Fig. 20); first three protarsomeres are dilated, the first being slightly narrower than the anterior tip of protibia; mesotibiae arched and metatibiae slightly arched.

Female
Antennae long, reaching the basal third of elytra, with antennomeres generally slightly longer and transverse (Table 1).Pronotum not so transverse; in dorsal view subrectangular, with side margins almost straight before maximum width; which is situated towards the third part of basal area (Fig. 15).Elytra elliptical and elongate, with notched and serrated apical areas.Sexual dimorphism very clear in pronotum, protibiae and apical part of elytra.Females are generally less robust and with less transverse pronotum than males.The uroventrites 7 th and 8 th , and spermatheca follows the overall model for the group.

Note
The great variability of this species led to the description of the subspecies zariquieyi Jeannel, 1936, later synonymized by Blas (1977), who observed that the differences correspond to intermediate forms and, moreover, the proximity of the different caves does not represent any real evidence of geographic isolation.

Biology and Ecology
Speonemadus escalerai is a subterranean species and, therefore, common in caves.It is mostly found in areas with small accumulations of bat guano, but also under stones or stalagmitic floors and in deep parts of caves (Blas 1989).Blas (1989), Giachino & Vailati (1993) and Fresneda et al. (2007) also pointed out its presence in the Superficial Subterranean Habitat (MSS) (sensu Giachino & Vailati 2010) in the karst area of Murcia.

Distribution
This species is distributed in the southeast Iberian Peninsula and colonizes caves of the provinces of Alicante, Murcia and Valencia in Spain (Fig. 32).

Speciation and biogeography of the S. escalerai-group
The genus Speonemadus is distributed in the Western Mediterranean area.It is known from Algeria, Spain, Italy, Morocco, Portugal, Tunisia and the south of France, including the Mediterranean islands of Sicily and the Balearics.
The synapomorphies of the escalerai-group are: male protibia with keels, females with the apex of the elytra notched and serrated (Giachino & Vailati 1993).The five species of this group are endemic to the Iberia Peninsula.
Scarce molecular data are reported for some species of Speonemadus (Fresneda et al. 2011): for S. angusticollis and S. bolivari, both of the escalerai-group, for S. clathratus, a widespread Iberian endemic, and for S. maroccanus, an ibero-Maghrebian species distributed in Morroco and in northeast Spain, the provinces of Cádiz, Granada and Málaga (Jeannel 1936;Coiffait 1954;Blas 1976Blas , 1977Blas , 1979Blas , 1981Blas , 1985Blas , 1989;;Bellés 1987;Fresneda et al. 2007Fresneda et al. , 2011;;Giachino & Vailati 1993;Tinaut 1998;Perreau 2000;Löbl & Smetana 2004;Salgado et al. 2008).In the phylogenetic analysis given by Fresneda et al. (2011, fig. 74), S. angusticollis and S. bolivari appear in the same clade, while S. clathratus is closer to the previously mentioned species than to S. maroccanus.This observation, together with the similarity and consistency of the morphological characters, including very similar aedeagi, led us to think that the diversification of the species of the escalerai-group was recent and probably related to the climatic changes of the Pleistocene.In this climate change scenario, an ancestor capable of inhabiting different habitats became isolated underground, through the progressive environmental aridity at the surface in the southern Mediterranean region, therefore leading to a reduction of gene flow and the existence of allopatry in isolated populations in the climatic shelter provided by the subterranean spaces.(Kraatz, 1870), S. bolivari (Jeannel, 1922), S. breuili (Jeannel, 1922) and S. escalerai (Uhagón, 1898).
The species of the escalerai-group and related species have a Betic-Rif distribution, where its origin might be located.There is some parallelism with the southern distribution of the Trechus fulvus-group (Carabidae), where multiple independent colonizations of the subterranean environment have been proposed based on the existence of troglomorphic species in clades that also include epigean species (Faille et al. 2014;Reboleira et al. 2009Reboleira et al. , 2010a)).
The karst of the Algarve, in southern Portugal, is now considered a hospot for a subterranean-adapted fauna (Reboleira 2012), with a considerable increase in the number of species described over the last decade.Most of them are clearly relicts.

Table 1 .
Mean measurements (in millimeters) of the antennomers of Speonemadus of the escaleraigroup (L) length; (W) width.