A review of Dendrocephalus ( Dendrocephalinus ) ( Crustacea : Anostraca ) with the first records of male-male anostracan aggressive competition

We present a review of Dendrocephalus (Dendrocephalinus) with an updated diagnosis for the subgenus and a key to all known species. We provide new records of Dendrocephalus alachua, which was previously supposed to be extinct, and we describe a new species, Dendrocephalus proeliator sp. nov., which is separated from all other species based on the form of the male frontal appendage. Dendrocephalus proeliator sp. nov. appears to be morphologically intermediate between D. alachua and D. lithaca. In addition, we provide conservation assessments for all four species in the subgenus, according to IUCN Red List standards. We also report for two species the first known examples of direct male-male agonistic behaviour and competition for access to areas frequented by receptive females.


Introduction
Dendrocephalus Daday, 1908 is a new world genus in the Thamnocephalidae, with two subgenera (Rogers 2006).Dendrocephalus s. str.ranges from Argentina, north through to Costa Rica, the Galápagos Islands, and Aruba, with 17 described species (Brendonck et al. 1990;Belk et al. 2002;Rogers 2013;Rogers et al. 2012;Rogers & Volcan 2016;Rabet et al. 2018).Rogers & Volcan (2016) provided keys to the species of this subgenus, and improved keys are in development (Rogers et al., in press).
The subgenus Dendrocephalinus is limited to the extreme southern United States of America (Georgia, Florida, and Texas), and has three described species (Rogers 2006(Rogers , 2013)).We review the subgenus, provide an updated key to the species, and discuss the conservation status of each.We present a new species of Dendrocephalinus from Florida and describe its remarkable male-male territorial aggressive behaviour.

Material and methods
Material was examined in museum collections or collected live from the wild, and cultured using the wild collections.Material of the new species was examined from the Florida Museum of Natural History, sent to the first author for identification.All preserved material was examined under a Wild M8 stereo dissection microscope.All drawings are freehand.Live adult specimens were collected using a dip net from the water column.The specimens are preserved in 70% ethyl alcohol, which was changed after 24 hours.Substrate from one of the localities was collected together with live eggs of the new species.
Cultures were started using techniques that simulate natural seasonal temperature and hydrologic cycles of the original collection habitat.A 1 l portion of the substrate was gently deposited in a plastic culture container (58 cm × 41 cm × 16 cm), and lightly sprayed with deionized water to prevent dust or debris and eggs from being displaced into the air during inundation.The substrate was initially inundated by adding 16 l of deionized water that had been warmed to 25°C.The culture was then gently stirred to break up substrate clumps, and gentle aeration via an air stone was added at one corner of the container.The culture was incubated at 25°C for 12 hours, then let come to room temperature.After 24 h, a second 16 l of 25°C deionized water was added to the culture container.The culture was maintained at room temperature under T12 fluorescent grow lights on a timed 12:12-h light:dark cycle.Cultures were fed 5 ml to 10 ml of a diet high calcium plant flake food (WorldWide Aquatics, Inc.) ground to a fine powder.In addition, 48 h after the second addition of water, 20 ml of a free swimming green alga culture (Selenastrum sp.) was added for additional food.

DCR
= author's own collection FLMNH = Florida Museum of Natural History, Florida, USA USNM = National Museum of Natural History, Washington, D.C., USA

Remarks
We suspect that this taxon may actually represent an independent genus, however molecular analyses will be required to demonstrate this.At this time we choose to conservatively maintain the taxon as a subgenus.

Remarks
This species is unique in that the 2A sub-branch (the apical branch) is absent, and sub-branch 2V and 1D are identical.The female shares the elongated brood pouch form with D. proeliator sp.nov.The only member of the subgenus occurring outside the extreme southeastern USA: all records are from southern, coastal Texas.All known locality substrates have 10-15% calcium carbonate and 2-8 mS/cm salinity (Rogers 2014b).

Remarks
The original description of the female has the brood pouch elongate, almost fusiform.In many, but not all, female specimens I have cultured, the brood pouch is abbreviated, lacking the free distal portion (Fig. 3E).This species is known from five unspecified collections in Alachua County, Florida, near Gainesville (Dexter 1953;Moore 1967; two unpublished records at USNM), and was suggested as possibly extinct, pending further surveys, even after many surveys were performed (Rogers, 2006).However, collecting efforts by W.W. Price yielded two new localities in the Tampa region.The Tampa locality is at least 180 km south of the unspecified type locality (a "barrow pit" (Dexter 1953) which has probably been destroyed (Rogers 2006)).For both new locations for this species the substrate has no measurable calcium carbonate and salinity 0 to ~2 mS/cm, as both locations are heavily vegetated, and have tree cover, with the substrate covered in vegetative debris (Rogers 2014b; this study).Similarly, both locations are less than a meter above the water table.
Dendrocephalus alachua co-occurred at both new locations with the smooth clam shrimp Lynceus gracilicornis Packard, 1871, and the spiny clam shrimp Eulimnadia cylindrova Belk, 1989. Both Dexter (1953) and Moore (1967) reported D. alachua co-occurring with the anostracan Streptocephalus sealii Ryder, 1879.However, this species was not present at either of the two new localities.Rogers (2006) conservatively designated D. alachua as CR A1 species (IUCN 2000) pending further surveys.But with these new collections this species meets the IUCN red list CR B1a,b species criteria (IUCN 2000), that the species is 'Critically Endangered', with an extent of occupancy less than 100 km 2 , and a projected decline in extent of occurrence.(Creaser, 1940) Fig. 1C, G

Remarks
This species is known from a set of rock outcrop pools on Stone Mountain in Georgia and has only been collected twice (Dexter 1953).A petition was made to list D. lithacus as endangered under the United States Federal Endangered Species Act.However, after extensive survey efforts no evidence of extant populations of D. lithacus was found (Federal Register May 27, 1998) and the petition was denied (Rogers 2006).Surveys made by the Georgia Department of Natural Resources at Stone Mountain and other granitic plutons in the area have so far yielded no new populations.This species is most likely extinct.The clam shrimp Eulimnadia graniticola Rogers et al., 2010

Diagnosis
Males are typical of the subgenus Dendrocephalinus, and readily identified in that the frontal appendage primary arms are each triramal, with branch 2A bearing an apical hamulate projection.The antenna-like appendage is extremely small and may be obscured.Females are not separable.

Etymology
The specific epithet 'proeliator' is Latin for 'warrior'.The gender is masculine.

Type locality
The type locality is Pond #107.1 at the Archbold Biological Station; a temporary pool in calcareous wet prairie.The substrate is Duette Sand, the pool fills to a maximum observed depth of 10 cm, and the subcircular basin is approximately 15 m across.These soils are calcareous and have a salinity of ~2 mS/ cm.The basin substrate has 100% vegetative cover, dominated by approximately 70% yellow hatpin (Syngonanthus flavidus (Michx.)Ruhland, 1903) and 30% Florida bluestem (Andropogon floridanus Scribn., 1896).A cluster of scrub oaks (possibly Quercus geminata Small, 1897) is in the center, providing shade and leaf litter.The surrounding area is scrubby flatwoods; a mixture of saw palmetto and shrubs with scattered pitch pine.

Female
Generally as typical for the genus (Fig. 2B).
Brood pouch.Fusiform, extending to proximal margin of postgenital abdominal segment IV.

Differential diagnosis
Of the four species of Dendrocephalus (Dendrocephalinus), males of D. proeliator sp.nov.are morphologically intermediate between D. lithacus (Fig. 1C) and D. alachua (Fig. 1B).These three species are easily differentiated from D. acacioidea (Fig. 1A) which lacks branch 2A in the frontal appendage.The three remaining species bear the lateral, broad surface of branch 2A with its rows of spiniform projections.Dendrocephalus proeliator sp.nov.bears three rami, as does D. alachua, and the three rami are superficially similar in overall structure between the two taxa.However, the hamulate distal projection of D. proeliator sp.nov. is absent in D. alachua.This hamulate distal projection is present in D. lithacus.Dendrocephalus proeliator sp.nov. is further separated from D. alachua in that branch 2V has eight total lateral lobes verses nine in D. alachua, and branch 2D is long, flagelliform and smooth in D. proeliator sp.nov.instead of vermiform and bearing rings of spines in D. alachua.Furthermore, branch 2D is proportionately much longer in D. proeliator sp.nov.than in D. alachua.Dendrocephalus alachua also has a distomedial spiniform projection at the apex of the second antennal distal antennomere, which is lacking in D. proeliator sp.nov.
Dendrocephalus lithacus lacks the branch 2D found in the other two species, and branch 2A is proportionately larger and more robust than the condition found in D. proeliator sp.nov.The hamulate projection of branch 2A in D. proeliator sp.nov. is approximately one fourth the total length of the branch, whereas in D. lithacus it is nearly half the branch length.Dendrocephalus lithacus also bears a row of fine spinulae on the second antenna proximal antennomere just basal to the frontal appendage, and a second row on the posteriolateral margin of the frontal appendage main trunk.These are lacking in D. proeliator sp.nov.and D. alachua.The second antennae distal antennomere medial margin bears spaced transverse grooves in D. lithacus versus a row of fine denticles in D. proeliator sp.nov.-Frontal appendage with sub-branch 2D with small scattered spines; sub-branch 2A lacking a chitinised, hamulate spine (Fig. 1B) ......... Dendrocephalus alachua (Dexter, 1953) (USA: Florida)

Discussion
The subgenus Dendrocephalinus was suggested to have split from Dendrocephalus sometime in the late Paleozoic (around 270 million years ago) when South America and North America were together at the equator, or at some point Dendrocephalinus was the result of founder Dendrocephalus populations invading North America (Rogers 2006).The fact that D. acacioidea is quite different in terms of frontal appendage morphology and is disjunct from the other three species, suggests that it may be basal to the other species.The strong similarity of gonopodal morphology leaves little doubt that it belongs in Dendrocephalinus.
Dendrocephalinus is smaller, more gracile, and the frontal appendage is greatly reduced and less elaborate than in Dendrocephalus s. str.Sexually mature adult preserved Dendrocephalus s. str.material in DCR's collection range from 10 to 16 mm total body length (from anterior margin of the head to posterior margin of the telson), whereas in Dendrocephalinus the range is from 4 to 9 mm.
The frontal appendage of Dendrocephalus s. str.has two ventral branches (1V, which is biramal, and 2V), a bi-or triramal dorsal branch (2D), and an apical branch that bears some form of a chitinised hook (2A).In Dendrocephalinus only the single ventral branch remains (1V), and it is of a single ramus that strongly resembles the 1V anterior sub-branch of Dendrocephalus s. str.The 2D branch, if present, is only one ramus, which either resembles 1V or is flagellar.The 2A, if present, is broad, and covered with chitinised papillae, and may or may not have the apical hook.
The elaborate male-male aggressive behaviour over hill topping sites is competition for access to receptive females.Anostracans live in seasonally astatic aquatic habitats with highly stochastic hydroperiods.This would suggest that aggressive interactions would be time ill spent; the limited hydroperiod would necessitate that the animals focus on reproduction.However, these species live in the Coastal Plain Anostracan Bioregion (Rogers 2014a), which frequently has long periods of precipitation, particularly in the summer months, from tropical storms and hurricanes.Thus, it is likely that these pools have prolonged hydroperiods providing time to engage in more competitive actions.
With the discovery of a new species in the subgenus, the existing key (Rogers & Hann 2016) is no longer useful.Thus, we provide an updated key to the subgenus here.