A New Species of Elpidium (Crustacea: Ostracoda: Limnocytheridae) from Brazil and a Morphological Phylogenetic Proposal for the Genus

© 2022 Academia Sinica, Taiwan Open Access A New Species of Elpidium (Crustacea: Ostracoda: Limnocytheridae) from Brazil and a Morphological Phylogenetic Proposal for the Genus Julia S. Pereira1,* , Carlos E. F. Rocha2, Ricardo L. Pinto3, and Marcio B. DaSilva1 1Universidade Federal da Paraíba (UFPB), Centro de Ciências Exatas e da Natureza, Departamento de Zoologia, Campus I, Castelo Branco, CEP 58051-900, João Pessoa/PB, Brasil. *Correspondence: E-mail: [email protected] (Pereira). E-mail: [email protected] (DaSilva) 2Universidade de São Paulo, Instituto de Biociências, Departamento de Zoologia, Rua do Matão, trav. 14, n°. 101, CEP 05508-090, São Paulo/SP, Brasil. E-mail: [email protected] (Rocha) 3Universidade de Brasília, Instituto de Geociências, Laboratório de Micropaleontologia, ICC-Ala Central, Subsolo ASS339/10, Campus Universitário Darcy Ribeiro, Asa Norte, CEP 70910-900, Brasília/DF, Brasil. E-mail: [email protected] (Pinto) Received 23 October 2021 / Accepted 13 April 2022 / Published 15 July 2022 Communicated by Ryuji Machida Elpidium species exclusively inhabiting confined and temporary environments, such as those of tankbromeliads, are a source of interesting and diverse studies on taxonomy, evolution and ecology, to name a few. However, despite its great diversity of species or potential for study, this genus (and other phytotelm members) has been poorly studied. In the last years, however, description of Elpidium species increased from six before 2013 to 11 today. This study is an effort to keep uncovering its great diversity and to go further in order to deeply understand the genus Elpidium. To this end, this study describes another species in the genus, Elpidium litoreum sp. nov., and proposes a phylogenetic reconstruction of it based on morphological characters. Our results point to the monophyly of Elpidium and puts Intrepidocythere ibipora as its sister-group. Although the phylogeny revealed some interesting relations, it also exposed some incongruities that ultimately demonstrate how superficial the current knowledge about the genus is. All these questions are discussed in detail. We see this work as at the same time an effort to better understand Elpidium and a stimulus to other researches to turn their attention to the historically neglected phytotelmata community. Key words: Limnocytheridae, Taxonomy, Evolution, Bromeliad fauna, Microcrustaceans. BACKGROUND Ostracods are microcrustaceans with an outstanding diversity in terms of described genera and species; they also inhabit an impressive variety of environments (Martens et al. 2008; Gusakov et al. 2021). The group has experienced a shift from marine to freshwater habitats several times over its evolutionary history. One of the most successful families in freshwater environments is the Limnocytheridae, with remarkable diversification and worldwide distribution (Park et al. 2002; Martens et al. 2008). Among limnocytherids, the genus Elpidium Müller, 1880 matchlessly adapted to living in bromeliad water tanks. Plants or plant structures capable of holding limited amounts of water—such as pitcher-plants, bamboos and tank-bromeliads—are called phytotelmata (Kitching 2001; Srivastava et al. 2004). Elpidium was first described by Müller (1880) with the publication of type-species Elpidium bromeliarum Müller, 1880, Citation: Pereira JS, Rocha CEF, Pinto RL, DaSilva MB. 2022. A new species of Elpidium (Crustacea: Ostracoda: Limnocytheridae) from Brazil and a morphological phylogenetic proposal for the genus. Zool Stud 61:27. doi:10.6620/ZS.2022.61-27. Zoological Studies 61:27 (2022) doi:10.6620/ZS.2022.61-27 1 © 2022 Academia Sinica, Taiwan from southern Brazil. Later on, Tressler (1941 1956) described E. maricaoensis (Tressler, 1941) and E. laesslei (Tressler, 1956), respectively, from Jamaica and Puerto Rico. Pinto and Sanguinetti (1962) compared the carapace morphology of several living and fossil genera of Timiriaseviinae, including Elpidium and proposed diagnostic features for each genus. In 1970, new material from southern Brazil allowed the proposition of a neotype and neoparatypes for E. bromeliarum by Pinto and Purper (1970), along with detailed redescriptions. Danielopol (1975) subsequently reported on three new species from Cuba but left them in open nomenclature. These three species were eventually formalized as E. inaequivalve Danielopol, 1981; E. purperae Danielopol, 1981; and E. pintoi Danielopol, 1981 (Colin and Danielopol 1981). For information about corrections in the specific epithet spellings and publication dates of the three latter species, see Meisch et al. (2019). Pinto and Jocqué (2013) also described E. merendonense Pinto and Jocqué, 2013 from Honduras. Interest in the evolutionary biology of Elpidium was advanced mainly by Little and Hebert (1996). Based on morphological and genetic aspects of Jamaican species, these authors suggested that bromeliads act as ecological islands, favoring high speciation and endemicity in Elpidium. The material used by Little and Hebert (1996) received formal taxonomical treatment in two publications. Danielopol et al. (2014) described E. martensi Danielopol, Pinto, Gross, Pereira and Riedl, 2014 and Pereira et al. (2019) added another three descriptions, E. littlei Pereira, Rocha and Pinto, 2019 E. heberti Pereira, Rocha and Pinto, 2019 and E. wolfi Pereira, Rocha and Pinto, 2019. Thus, the genus currently comprises 11 valid species. However, the sampling effort for phytotelmata inhabitants is still scarce (Jocqué et al. 2013). Consequently, this figure possibly represents an underestimation of the actual Elpidium diversity. In terms of ecology, in the original discovery of Elpidium, Müller (1880) proposed a probable phoretic behavior. Indeed, Lopez et al. (1999 2002 2005) later demonstrated that Elpidium species disperse between bromeliads by using amphibians as phoretic vectors. In the present work, we describe a new species of Elpidium and present a morphological phylogenetic analysis of the genus. We see this study as an effort to do a more pluridisciplinary study, following the proposition of Danielopol et al. (2014) to integrae different biological aspects that can ultimately lead us to a deeper understanding of Elpidium. MATERIALS AND METHODS Specimens were collected from unidentified tankbromeliads in Arraial do Cabo, Rio de Janeiro, Brazil. Two populations of the new species were found, one from sandy beaches in Praia Grande and another one from a rocky shore in Praia do Forno. All illustrations and type-series presented below were derived from the material collected in Praia Grande. Water samples were collected from bromeliad tanks with the aid of a pipette and taken to the laboratory for ostracod sorting. Specimens were preserved in alcohol 90%. Adult specimens were dissected under a stereomicroscope; soft parts were mounted in permanent slides using CMC-9AF mounting medium and valves were stored dry in micropaleontological slides. Appendages were drawn under the microscope with the aid of a camera lucida. Both valves of dissected specimens and closed carapaces of males and females were photographed using scanning electron microscopy (SEM). All material used for the description is deposited in the crustacean collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP). Higher taxonomy follows Horne et al. (2002) and Danielopol et al. (2014). For the phylogenetic analysis, 77 morphological characters were erected (Table S1) and organized in two different matrices, one for continuous (Table S2) and another for discrete characters (Table S3). With the software Mesquite 3.6 (Maddison and Maddison 2018), the two matrices were generated and subsequently exported to a text (.txt) file. Both text files were then manually combined into one mixed matrix, composed of 16 species and 77 characters. Three of the 16 species were used as outgroup—Cytheridella sp., Gomphocythere huwi Martens, 2003 and Thaicythere srisumonae Savatenalinton et al., 2008—while the remaining 13 species were treated as the internal group: all species of Elpidium and Intrepidocythere ibipora Pinto et al., 2008, since this monospecific genus is morphologically very similar to Elpidium. Of the 77 morphological characters, 35 were extracted from the carapace and 42 from appendages, from which 11 were extracted specifically from sexual appendages. Five of these 77 morphological characters were continuous, and measurements were made with the software Zeiss Axiovision 4.8 using the holotype and allotype of each species. Whenever possible, new dissections of fresh material or available dissected specimens were used for direct verification of character states. The material used is detailed below. In the outgroup, we directly verified the characters of Cytheridella from available material sampled in page 2 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Lake Paranoá, Brasíla, Brazil (taxonomy of this species will be presented elsewhere). We relied on the original descriptions for Gomphocythere huwi and Thaicythere srisumonae, since they present thorough descriptions and illustrations. In the internal group, both the original description and direct examination of dissected specimens were used for I. ibipora. Within Elpidium, only published descriptions were used for E. inaequivalve, E. purperae and E. pintoi and for the type species of the genus, E. bromeliarum. For E. maricaoensis, E. merendonense and E. martensi, original descriptions and stored material were used. Concerning E. laesslei, E. wolfi, E. littlei and E. heberti, we directly examined the material sampled by Little and Hebert (1996) and re-described by Pereira et al. (2019). All the sources used for each species are fully detailed in table 1. The combined matrix of discrete and continuous characters was analyzed with the phylogenetic software Tree analysis using New Technology (TNT) (Goloboff and Catalano 2016). The analysis was performed using the exact solution algorithm “Implicit Enumeration”, applying the same weight to all characters. Characters 13, 19, 21–24, 38 and 72 were treated as additive, remaining characters were all non-additive. Characters 1 to 5 were analyzed without a priori discretization, following Goloboff et al. (2006). No consensus method was applied since a single most parsimonious tree was found (see RESULTS and DISCUSSION). RESULTS TAXONOMY Class Ostracoda Latreille, 1802 Subclass Podocopa Sars, 1866 Order Podocopida Sars, 1866 Suborder Cytherocopina Baird, 1850 Superfamily Cytheroidea Baird, 1850 Family Limnocytheridae Sars, 1925 Subfamily Timiriaseviinae Mandelstam, 1960 Tribe Timiriaseviini Mandelstam, 1960 Genus Elpidium Müller, 1880 Type species. Elpidium bromeliarum Müller, 1880. Table 1. Source of information used for each species for the character list and matrix Taxa Source of information Gomphocythere huwi Original description (Martens 2003). Thaicythere sirsumonae Original description (Savatenalinton et al. 2008). Cytheridella sp. Sample available (collected by Ricardo L. Pinto from Lake Paranoá, Brasília, Brazil). Intrepidocythere ibipora Original description (Pinto et al. 2008). Material stored at Museu de Zoologia da Universidade de São Paulo (MZUSP 18479, MZUSP 18480). Elpidium bromeliarum Original description (Müller 1880). Description of the neotypical series (Pinto and Purper 1970). Description of the ontogenetic series (Pereira et al. 2017). Elpidium maricaoensis Original description (Tressler 1941). Material stored at National Museum of Natural History, Smithsonian Institute (USNM 80029). Elpidium laesslei Re-description (Pereira et al. 2019). Sample available (collected by Little and Hebert 1996). Elpidium inaequivalvis Original description (Danielopol 1975). Elpidium pintoi Original description (Danielopol 1975). Elpidium purperae Original description (Danielopol 1975). Elpidium merendonense Original description (Pinto and Jocqué 2013). Material stored at Museu de Zoologia da Universidade de São Paulo (MZUSP 29072, MZUSP 29073). Elpidium martensi Original description (Danielopol et al. 2014). Material at stored at Museu de Zoologia da Universidade de São Paulo (MZUSP 32812, MZUSP 32813). Material collected by Little and Hebert (1996). Elpidium littlei Original description (Pereira et al. 2019). Material collected by Little and Hebert (1996). Elpidium heberti Original description (Pereira et al. 2019). Material collected by Little and Hebert (1996). Elpidium wolfi Original description (Pereira et al. 2019). Material provided by Prof. Dr. Wilhelm Foissner (University of Salzburg, Austria). Elpidium litoreum sp. nov. Material collected by Julia S. Pereira, Danielly G. Oliveira and Dariane I. D. Schneider (here described). page 3 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Species included in the genus: Elpidium maricaoensis (Tressler, 1941); Elpidium laesslei (Tressler, 1956); Elpidium inaequivalve Danielopol, 1981; Elpidium pintoi Danielopol, 1981; Elpidium purperae Danielopol, 1981; Elpidium merendonense Pinto & Jocqué, 2013; Elpidium martensi Danielopol et al., 2014; Elpidium littlei Pereira et al., 2019; Elpidium heberti Pereira et al., 2019; Elpidium wolfi Pereira et al., 2019; Elpidium litoreum sp. nov. Diagnosis (modified after Pinto and Jocqué 2013): Medium to large sized carapace, generally with subtle ornamentation marked by minute individual or grouped foveolae (with the exception of Elpidium laesslei). Brownish color, varying from light to dark. Width larger than height, ventral surface flat. Bisexual, with sexual dimorphism on both carapace and appendages varying from subtle to outstanding, but always present. Males with greatest width usually at mid-length; females broader than males posteriorly due to the existence of a brooding chamber, and greatest width displaced posteriorly. Antennula 5-segmented. First segment bearing dorso-apical expansion set with pseudochaetae. Antenna with 2 biserrate claws and 1 pectinate claw in males and 3 bisserrate claws in females; hyaline formation on terminal segment in both males and females. Maxillula with 2 spatulate claws in each second and third endites. Hemipenis greatly sclerotized; caudal ramus reduced to a pair of setae; copulatory process usually a hook-like structure with ejaculatory glans and ducts united or separated; distal lobe with dorsal seta, both varying in shape and size; lower ramus present and varying in shape; upper ramus absent. Females with abdomen rounded, bearing a stiff dorsal spine; caudal ramus reduced as in males; genital operculum sclerotized. Elpidium litoreum sp. nov. Pereira, Rocha, Pinto and DaSilva (Figs. 1–6) urn:lsid:zoobank.org:act:70B73BBB-35FB-4B16-A347A41BE2B7F312 Diagnosis: Small-sized Elpidium, markedly elongated (length/width ratio = 1.4; length/height ratio = 1.9–2.0). Brownish carapace surface with sparse setae, normal pore canals and subtle ornamentation, represented by minute individual foveolae. In dorsal and ventral views, carapace symmetric. Ventral surface flat. In right lateral view, left valve overlaps right one in all margins; dorsal margin slightly arched, straight on the central portion; ventral margin arched, not straight; external antero-ventral flange well marked. Sexual dimorphism outstanding: in dorsal and ventral views posterior margin rounded in males while truncate in females. Hemipenis with left and right distal lobes elongated (distal lobe basis width/ distal lobe length ratio = 0.4), but asymmetric in shape: left one subquadrate, right one with curved apex; vestigial digital expansion present medially. Copulatory process a simple short hook-like ejaculatory duct. Lower ramus with broad basis, tapering towards lancet-shaped apex. Type material: Holotype: a dissected ♂ (MZUSP 38804) with valves dried and coated for scanning electron microscopy stored in a micropaleontological slide and appendages mounted in a sealed slide with glycerin. Allotype: a dissected ♀ (MZUSP 38805) stored like the holotype. Paratypes: a ♂ (MZUSP 38820) and 2 ♀ (MZUSP 38807, MZUSP 38818) dissected and stored like the holotype; 3 ♂ (MZUSP 38806, MZUSP 38808, MZUSP 38815) dissected with appendages mounted in a sealed slide with glycerin; 5 ♂ (MZUSP 38812, MZUSP 38813, MZUSP 38814, MZUSP 38817, MZUSP 38819) and 4 ♀ (MZUSP 38809, MZUSP 38810, MZUSP 38811, MZUSP 38816) dried and coated for scanning electron microscopy stored in micropaleontological slides; about 136 ♂ and 150 ♀ (MZUSP 38821) kept whole in a vial with 70% alcohol. Type locality: Tank-bromeliads from Praia Grande, Arraial do Cabo, Rio de Janeiro, Brazil. Approximate geographical coordinates: 22°98'S 42°03'W. Material collected in 11.x.2013 by Julia S. Pereira, Danielly G. Oliveira and Dariane I. D. Schneider. Additional material: 3 ♂ (MZUSP 38822, MZUSP 38830, MZUSP 38831) and 2 ♀ (MZUSP 38823, MZUSP 38824) dissected and stored like the holotype; 3 ♂ (MZUSP 38825, MZUSP 38826, MZUSP 38828) and 2 ♀ (MZUSP 38827, MZUSP 38829) dried and coated for scanning electron microscopy stored in micropaleontological slides; 5 ♀ (MZUSP 38832) kept whole in a vial with 70% alcohol. Locality: Tank-bromeliads from a rocky shore in Praia do Forno, Arraial do Cabo, Rio de Janeiro, Brazil. Approximate geographical coordinates: 22°57'S 42°00'W. Material collected in 11.x.2013 by Julia S. Pereira, Danielly G. Oliveira and Dariane I. D. Schneider. Derivation of name: The specific epithet “litoreum” refers to the occurrence of the species (and its host bromeliads) on the beaches of Arraial do Cabo, Rio de Janeiro, Brazil. It derives from the Latin adjective “litoreus”, meaning from the beach, coastal. Description of the male: Carapace (Fig. 1A–I). Small-sized Elpidium (length of holotype = 629.8 µm), carapace elongated (length/width ratio = 1.4; length/ height ratio = 1.9). Color varying from light to dark brown. Subtle ornamentation numerous and minute individual foveolae. Normal pore canals and sparse page 4 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Fig. 1. Elpidium litoreum sp. nov., ♂, carapace. A, dorsal view (MZUSP 38812); B, dorsal view, detail of anterior region (MZUSP 38812); C, dorsal view, detail of posterior region (MZUSP 38812); D, ventral view (MZUSP 38813); E, ventral view, detail of anterior region (MZUSP 38813); F, ventral view, detail of posterior region (MZUSP 38813); G, right lateral view (MZUSP 38812); H, right lateral view, detail of anterior region (MZUSP 38812); I, right lateral view, detail of posterior region (MZUSP 38812); J, left valve, internal view (MZUSP 38819); K, left valve, internal view, detail of anterior region (MZUSP 38819); L, left valve, internal view, detail of posterior region (MZUSP 38819); M, right valve, internal view (MZUSP 38831); N, right valve, internal view, detail of anterior region (MZUSP 38831); O, right valve, internal view, detail of posterior region (MZUSP 38831). Scale bars: A, D, J, M = 100 µm; B, C, E, F, H, N, O = 20 µm; G = 30 µm; I = 10 µm; K, L = 50 µm. page 5 of 17 Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan setae present. In dorsal and ventral views, posterior region slightly broad; posterior margin rounded, not pointed; ventral surface flat. In right lateral view, left valve overlaps right valve on all margins; dorsal margin slightly arched, straight on the central portion; ventral margin arched, not straight; external antero-ventral flange outstanding. Left valve (Fig. 1J–L): Flange present in anterior margin, absent in ventral and posterior margins. Selvage well marked in anterior margin; bow funnel-shaped structure in oral region. Calcified inner lamella broad in anterior and posterior regions; inner list well marked on anterior calcified inner lamella and subtle with a row of minute pseudochaetae on posterior calcified inner lamella. Vestibule broad in anterior and posterior regions. Adductor muscle scars 4-stacked spots on valve anterior third. Right valve (Fig. 1M–O): Flange present in anterior, ventral and posterior margins, the latter one with sparse setae. Selvage well marked in anterior, ventral and posterior margins; bow funnel-shaped structure in oral region, interrupting flange. Calcified inner lamella broad in anterior and posterior regions; inner list subtle on anterior calcified inner lamella and well-marked on posterior calcified inner lamella. Vestibule broad in anterior and posterior regions. Adductor muscle scars 4-stacked spots on valve anterior third. Hinge about 2/3 of the dorsal margin extension; cardinal bar with 2 proto-teeth: posterior one more developed; bar and proto-teeth with very small crenulated ornamentation, visible only under high magnification. Antennula (Figs. 2A, 3A - represented by female specimen): 5-segmented. First segment relatively long bearing sub-apical expansion with a tuft of tiny pseudochaetae. Second segment the longest, with a single plumose seta in ventro-proximal position reaching fourth segment. Third segment square-shaped with a unique serrate seta in dorso-apical position that reaches fourth segment at about mid-length. Fourth segment bigger in length than in width and partially subdivided slightly before mid-length; medially with 2 dorsal sub-equal serrate setae and one ventral serrate seta; apically with a very long ventral serrate seta and 3 dorsal serrate setae: 1 short and 2 long, sub-equal length. Fifth segment (terminal) with 3 serrate and thin setae, 1 long and 2 equally short, plus an aesthetasc (Ya). Third, fourth and fifth segments with a row of pseudochaetae, covering whole or part of their apical portions. Antenna (Figs. 2B, 3B, E, F, 3C, D - represented by female specimen): Protopodite 2-segmented; coxa ring-shaped and basis long and arched, dorsally with 2 rows of tiny pseudochaetae and ventrally a triangular-shaped group of pseudochaetae. Endopodite 3-segmented. First endopodal segment relatively short, with very long serrate ventro-apical seta reaching apical portion of second endopodal segment; a group of pseudochaetae ventrally and 3 separated groups of long pseudochaetae dorsally, with each group reaching the next one in length. Second endopodal segment the longest, with a hardly visible vestigial seta apically, 2 sub-apical setae, 1 half as long as the other, dorsally and ventrally, about mid-length, a serrate seta and an aesthetasc (Y). Third segment (terminal) with 3 claws of sub-equal length, 2 serrate and 1 pectinate with a very strong row of denticles, besides tiny seta and hyaline formation. Exopodite very long and arched spinneret seta and vestigial basal seta. Mandible (Fig. 2F–H, represented by female specimen, 3G, H): Coxa internally with 8 strong teeth, modified X1 seta (spoon-shaped) and long plumose seta plus 2 interdental setae (X2 and X3), 3 interdental spines and sub-apical plumose seta. Palp 4-segmented: basis and 3 endopodal segments; basis with 2 setae in subapical position and respiratory plate (the exopodite) with 3 long setae and 1 short, reflexed seta, all with tiny setulae; first endopodal segment with 2 apical setae, 1 half as long as the other; second endopodal segment with 4 apical setae, 2 long and 2 short; third endopodal segment (terminal) with 3 setae, 2 thin and similar in length and 1 larger and longer than the other 2. Maxillula (Fig. 2E): Bearing 3 endites. First one with 3 slender setae, approximately equally long; second and third endites with 2 spatulate claws and 3 smooth and slender setae each. Palp with about 4 rows of tiny pseudochaetae medially-positioned and 2 long plumose setae plus vestigial seta on apical portion. Respiratory plate (exopodite) well developed, with minute spines centrally and 16 rays plus 1 reflexed seta, all plumose. First thoracic limb (Figs. 4A, 5A–D): Basis long and slightly arched with several rows of tiny pseudochaetae. Dorsal margin with medium-size plumose seta plus 2 short apical pappose setae wrapped in their basal portion by an expansion of segment. Exopodite a long and plumose seta. Endopodite 3 elongated segments. First segment the longest one, with several long pseudochaetae in both sides and one, strong biserrate seta, slightly shorter than second segment length; second segment devoid of setae; third segment (terminal) with strong and arched claw, slightly biserrate on its end and with tiny vestigial seta and row of pseudochaetae on its basis. All endopodal segments with rows of pseudochaetae in their apical and lateral portions. Second thoracic limb (Figs. 4B, 5E–G): Similar to first thoracic limb in general shape, but longer. Basis with only 1 plumose seta on its apical portion, also page 6 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Fig. 2. Elpidium litoreum sp. nov., A, B, E, ♂, C, D, F–H, ♀, appendages. A, antennula (MZUSP 38815); B, antenna (MZUSP 38815); C, antenna (MZUSP 38818); D, antenna, terminal segment (MZUSP 38805); E, maxillula (MZUSP 38815); F, mandible, coxa (MZUSP 38805); G, mandible, basis (MZUSP 38805); H, mandible, respiratory plate (MZUSP 38805). Scale bars: A–C = 0.05 mm; D–H = 0.01 mm. page 7 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Fig. 3. Elpidium litoreum sp. nov., A, C, D, ♀, B, E–H, ♂, appendages. A, antennula, detail of first segment, arrows point to the sub-apical expansion structure and to the two pseudochaetae rows (MZUSP 38816); B, antenna, detail of first protopodite segment (coxa), arrow points to the pseudochaetae row (MZUSP 38817); C, antenna, detail of second protopodite segment, arrows point to the numerous pseudochaetae rows (MZUSP 38816); D, antenna, first endopodal segment, arrows point to the three pseudochaetae groups (MZUSP 38816); E, antenna, second endopodal segment showing the aesthetasc (MZUSP 38817); F, antenna, portion of second endopodal segment and third endopodal segment, arrow points to the tiny vestigial seta (MZUSP 38817); G, mandible, portion of coxa (MZUSP 38817; H, mandible, portion of coxa, arrows point to the tiny spines (MZUSP 38817). Scale bars: A, D, E, H = 2 µm; B, C, F, G = 10 µm. page 8 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan wrapped by a segment expansion, but incompletely. Biserrate seta of the first endopodal segment and second segment equally long. Third segment (terminal) with strong apical claw, slightly longer and more arched than claw present on first thoracic limb terminal segment. Third thoracic limb (Figs. 4C, 5H–J): Basis with 3 setae: 2 slender setae dorsally (1 in medial and 1, pappose, in apical position) plus 1 plumose exopodial seta ventrally in medio-proximal position. All 3 endopodal segments with length greater than width and their total lengths greater than in the first and second thoracic limbs. First segment with a unique biserrate seta equal in length to second segment and slightly more slender than its homologous structure in first and second thoracic limbs; second segment with transversal row of pseudochaetae on its lateral portion (structure absent from first and second thoracic limbs) and without seta; third segment (terminal) with very long and thin claw with vestigial seta on its basis. This latter structure biserrate as in first and second thoracic limbs, but not only in its end but in approximately 2/3 of its length. All 3 endopodal segments with rows of pseudochaetae in their apical and lateral portions, longer than those on first and second thoracic limbs. Hemipenis (Figs. 4D, 5K–M): Large and sclerotized muscular body with copulatory complex (copulatory process and distal lobe) and furcal lobe as main structures. Furcal lobe with 2 pairs of mediumsized setae with numerous pseudochaetae. Distal lobe asymmetric; left one subquadrate, slightly longer than Fig. 4. Elpidium litoreum sp. nov., A–D, ♂, E, ♀, appendages. A, first thoracic limb (MZUSP 38815); B, second thoracic limb (MZUSP 38815); C, third thoracic limb (MZUSP 38815); D, hemipenis (MZUSP 38815); E, abdomen (MZUSP38805). Scale bars: A–E = 0.05 mm. page 9 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan S. Pereira, Ricardo L. Pinto and Marcio B. DaSilva contributed to the phylogenetic analyses. Competing interests: Julia S. Pereira, Carlos E. F. da Rocha, Ricardo L. Pinto and Marcio B. DaSilva declare that they have no conflict of interest. Availability of data and materials: List of morphological characters and matrix of both continuous and discrete characters are available in supplementary materials. Consent for publication: Not applicable. Ethics approval consent to participate: Not applicable. REFERENCES Colin JP, Danielopol DL. 1981. Sur la morphologie, la systématique, la biogéographie et l’évolution des ostracodes Timiriaseviinae (Limnocytheridae). Paleobiol Cont XI:1–51. Danielopol DL. 1975. Remarques sur la diversification morphologique de trois especes d’Elpidium (Ostracoda) a Cuba. Bull Am Paleontol 65:47–60. Danielopol DL, Pinto RL, Gross M, Pereira JS, Riedl N. 2014. On the evolutionary biology of Elpidium ostracods (Limnocytheridae, Timiriaseviinae): a proposal for pluridisciplinary studies. GeoEco-Marina 20:87–129. Goloboff PA, Catalano SA. 2016. TNT version 1.5, including a full implementation of phylogenetic morphometrics. Cladistics 32:221–238. doi:10.1111/cla.12160. Goloboff PA, Mattoni CI, Quinteros AS. 2006. Continuous characters analyzed as such. Cladistics 22:589–601. doi:10.1111/j.10960031.2006.00122.x. Gusakov VA, Makhutova ON, Gladyshev MI, Golovatyuk LV, Zinchenko TD. 2021. Ecological role of Cyprideis torosa and Heterocypris salina (Crustacea, Ostracoda) in saline rivers of the Lake Elton basin: abundance, biomass, production, fatty acids. Zool Stud 60:53. doi:10.6620/ZS.2021.60-53. Horne DJ, Cohen A, Martens K. 2002. Taxonomy, morphology and biology of quaternary and living Ostracoda. In: Holmes JA, Chivas AR (eds) The Ostracoda applications in Quaternary research. American Geophysical Union, Washington, DC, USA. Jocqué M, Fiers F, Romero M, Martens K. 2013. Crustacea in phytotelmata: a global overview. J Crustac Biol 33:451–460. doi:10.1163/1937240X-00002161. Karanovic I, Humphreys WF. 2014. Phylogeny and diversity of Timiriaseviinae ostracods (Podocopida, Cytheroidea) with description of one new species from arid Western Australia. Syst Biodivers 12:93–110. doi:10.1080/14772000.2014.882870. Kitching RL. 2001. Food webs in phytotelmata: “bottom-up” and “top-down” explanations for community structure. Annu Rev Entomol 46:729–760. doi:10.1146/annurev.ento.46.1.729. Külköylüoglu O, Akdemir D, Yavuzatmaca M, Yilmaz O. 2015. A checklist of recent non-marine Ostracoda (Crustacea) of Turkey with three new records. Rev Hydrobiol 8:77–90. Little TJ, Hebert PDN. 1996. Endemism and ecological islands: the ostracods from Jamaican bromeliads. Freshw Biol 36:327–338. Lopez LCS, Filizola B, Deiss I, Rios RI. 2005. Phoretic behaviour of bromeliad annelids (Dero) and ostracods (Elpidium) using frogs and lizards as dispersal vectors. Hydrobiologia 549:15–22. doi:10.1007/s10750-005-1701-4. Lopez LCS, Gonçalves DA, Mantovani A, Rios RI. 2002. Bromeliad ostracods pass through amphibian (Scinaxax perpusillus) and mammalian guts alive. Hydrobiologia 485:209–211. doi:10.1023/A:1021315223774. Lopez LCS, Rodrigues PJFP, Rios RI. 1999. Frogs and Snakes as Phoretic Dispersal Agents of Bromeliad Ostracods (Limnocytheridae: Elpidium) and Annelids (Naididae: Dero). Biotropica 31:705–708. doi:10.1111/j.1744-7429.1999.tb00421. x. Maddison WP, Maddison DR. 2018. Mesquite: a modular system for evolutionary analysis. Version 3.6. Available at: http://www. mesquiteproject.org. Martens K. 2003. On the evolution of Gomphocythere (Crustacea, Ostracoda) in Lake Nyassa/ Malawi (East Africa), with the description of 5 new species. Hydrobiologia 497:121–144. doi:10.1023/A:1025417822555. Martens K, Savatenalinton S. 2011. A subjective checklist of the Recent, free-living, non-marine Ostracoda (Crustacea). Zootaxa 2855:1–79. doi:10.11646/zootaxa.2855.1.1. Martens K, Schön I, Meisch C, Horne DJ. 2008. Global diversity of ostracods (Ostracoda, Crustacea) in freshwater. Hydrobiologia 595:185–193. doi:10.1007/s10750-007-9245-4. Meisch C, Mary-Sasal N, Colin JP, Wouters K. 2007. Freshwater Ostracoda (Crustacea) collected from the islands of Futuna and Wallis, Pacific Ocean, with a check-list of the non-marine Ostracoda of the Pacific Islands. Bull la Société des Nat Luxemb 108:89–103. Meisch C, Smith RJ, Martens K. 2019. A subjective global checklist of the extant non-marine Ostracoda (Crustacea). European Journal of Taxonomy 492:1–135. doi:10.5852/ejt.2019.492. Müller F. 1880. Descrição do Elpidium bromeliarum, crustaceo da familia dos cytherideos. Arch do Mus Nac do Rio Janeiro IV:27– 35. Park LE, Martens K, Cohen AS. 2002. Phylogenetic Relationships of Gomphocythere (Ostracoda) in Lake Tanganyika, East Africa. J Crustac Biol 22:15–27. doi:10.1163/20021975-99990205. Pereira JS, Rocha CEF, Pinto RL. 2019. On three new species of Elpidium Müller, 1880 and the re-description of Elpidium laesslei (Tressler, 1956) from Jamaica (Ostracoda: Podocopida: Limnocytheridae). Zootaxa 4604:103–140. doi:10.11646/ zootaxa.4604.1.4. Pereira JS, Schneider DID, da Rocha CEF, Pinto RL. 2017. Carapace ontogeny of the bromeliad dwelling ostracod Elpidium bromeliarum Müller, 1880 (Crustacea: Ostracoda). J Nat Hist 51:2185–2196. doi:10.1080/00222933.2017.1360529. Pinto ID, Purper I. 1970. A Neotype for Elpidium bromeliarum Müller, 1880 (type species for the genus) and a revision of the genus Elpidium (Ostracoda). Esc Geol Porto Alegre 19:1–23. Pinto ID, Sanguinetti YT. 1962. A complete revision of the genera Bisulcocypris and Theriosynoecum (Ostracoda) with the world geographical and stratigraphical distribution (including Metacypris, Elpidium, Gomphycythere and Cytheridella). Publicação Especial, Ecola de Geologia, Universidade do Rio Grande do Sul 4:1–165. Pinto RL, Jocqué M. 2013. A new species of Elpidium (Crustacea, Ostracoda) from bromeliads in Cusuco National Park, Honduras. Zookeys 313:45–59. doi:10.3897/zookeys.313.4904. Pinto RL, Rocha CEF, Martens K. 2008. On the first terrestrial ostracod of the Superfamily Cytheroidea (Crustacea, Ostracoda): Description of Intrepidocythere ibipora n. gen. n. sp. from forest leaf litter in São Paulo State, Brazil. Zootaxa 1828:29–42. doi:10.11646/zootaxa.1828.1.3. page 16 of 17Zoological Studies 61:27 (2022) © 2022 Academia Sinica, Taiwan Sabagh LT, Dias RJP, Branco CWC, Rocha CFD. 2011. News records of phoresy and hyperphoresy among treefrogs, ostracods, and ciliates in bromeliad of Atlantic forest. Biodivers Conserv 20:1837–1841. doi:10.1007/s10531-011-0050-z. Savatenalinton S, Borgonie G, Martens K. 2008. On Thaicythere srisumonae n. gen., n. sp. (Ostracoda) from Thailand, with notes on the phylogenyof the subfamily Timiriaseviinae Mandelstam, 1960. Crustaceana 81:411–432. doi:10.1163/156854008783797543. Scharf B, Meisch C. 2014. New records of nonmarine Ostracoda (Crustacea) from Gran Canaria (Canary Islands), with an updated checklist of the nonmarine Ostracoda of the Canary Islands. Bull la Société des Nat Luxemb 115:259–270. Sidorov DA, Semenchenko KA. 2012. New records of freshwater ostracods (Crustacea) from the Far East of Russia, with a checklist of recent freshwater ostracods of the region. Arthropoda Sel 21:227–234. doi:10.15298/arthsel.21.3.04. Srivastava DS, Kolasa J, Bengtsson J, Gonzalez A, Lawler SP, Miller TE, Munguia P, Romanuk T, Schneider DC, Trzcinski MK. 2004. Are natural microcosms useful model systems for ecology? Trends Ecol Evol 19:379–384. doi:10.1016/j.tree.2004.04.010. Tressler WL. 1941. Ostracoda from Puerto Rican bromeliads. J Washingt Acad Sci 31:263–269. Tressler WL. 1956. Ostracoda from bromeliads in Jamaica and Florida. J Washingt Acad Sci 46:333–336. Supplementary Materials Table S1. List of characters. (download) Table S2. Morphological matrix of continuous characters. (download) Table S3. Morphological matrix of discrete characters. (download) page 17 of 17Zoological Studies 61:27 (2022)