Caridina biyiga sp. nov., a new freshwater shrimp (Crustacea: Decapoda: Atyidae) from Leichhardt Springs, Kakadu National Park, Australia, based on morphological and molecular data, with a preliminary illustrated key to Northern Territory Caridina

Caridina biyiga sp. nov. is described from Leichhardt Springs, Kakadu National Park, Northern Territory, Australia, based on morphological and molecular data. The new species is highly distinctive among its congeners in having the dactylus of pereiopod 5 similar to the dactyli of pereiopods 3–4 and lacking the comb-like row of spiniform setae typical of the genus. The branchial formula is also distinctive for the genus in lacking an arthrobranch at the base of pereiopod 1 and in having a vestigial epipod on maxilliped 1. Despite the distinctive morphology of the new species, the molecular data did not provide strong support for the recognition of a new genus. Phylogenetic analyses of Australian Caridina using the mitochondrial 16S rDNA gene fragment placed the new species in a well-supported clade containing at least one typical species of Caridina. Within this clade, referred to as the ‘thermophila’ group, Kimura 2-parameter (K2P) genetic distances of 16S rDNA between the new species and sister taxa ranged from 5.1–6.0%. Analyses using the mitochondrial 3’ COI gene fragment from species of the ‘thermophila’ group yielded K2P genetic distances between the new species and its sister taxa ranging from 10.4–15.1%. A preliminary illustrated key to Northern Territory Caridina is provided. The conservation significance of Leichhardt Springs and its aquatic fauna are also briefly discussed.


Introduction
first reported Caridina from the Northern Territory in a paper discussing the zoogeography of Australian freshwater decapod Crustacea. Subsequently, Smith & Williams (1982) recorded C. wilkinsi (Calman, 1926) from Fogg Dam, Humpty Doo and near Jabiru in Kakadu National Park. Bruce (1983) reported C. gracilirostris De Man, 1892 (= Caridina 'sp. Gulf1' this study;sensu Page et al. 2007a) from Darwin River Dam. In a checklist of the Decapoda of Australian inland waters, Horwitz (1995) also listed C. typus H. Milne Edwards, 1837 and C. serratirostris (= Caridina 'sp. WA4' this study; sensu Page et al. 2007a) from the Northern Territory.
The most detailed treatment of the Northern Territory fauna to date was an extensive molecular phylogenetic study of Australian Caridina by Page et al. (2007a). Three undescribed species were reported from the Northern Territory: Caridina 'sp. NT 1' from Melville Island; Caridina 'sp. NT nilotica' from Bamboo Creek, Holmes Jungle and the Roper River at Roper River Bar; and Caridina 'sp. Gulf1' from Coomalie Creek (the same species previously listed from the Northern Territory as C. gracilirostris by Horwitz 1995 also recorded C. 'sp. NT nilotica' from the Blackmore River in the Northern Territory, as well as C. 'sp. WA4'. This last Genomic DNA was amplified from tail muscle tissue and sequenced in both directions as per Page et al. (2008c). The following primer combinations were used; 16S: 16S-F-Car and 16S-R-Car (von Rintelen et al. 2007); 3' COI: CDC0.La (Page et al. 2005b) and COIa-H (Palumbi et al. 1991); 5' COI: LCO-1490and HCO-2198(Folmer et al. 1994; 28S: 28S-F-Cru and 28S-R-Met (von Rintelen et al. 2012). For comparative purposes, 12 additional Australian atyid species were also sequenced for 16S using the primer combination above or 16S-ar (Palumbi et al. 1991) and 16S-R1-Car (von Rintelen et al. 2007) (see Table 2 for sampling site details for specimens sequenced in this study). A specimen of Caridina thermophila was also sequenced for 3' COI using CRCOI-F and CRCOI-F (Cook et al. 2008).
As a quick preliminary scan for any closely related, Indo-West Pacific species, sequences of the four gene fragments from the Leichhardt Springs specimens were compared to all available DNA sequences on GenBank (http:// blast. ncbi.nlm.nih.gov/) as of 12 June, 2019 and the 5' COI sequence compared to the BOLD database (http://www. boldsystems.org) on the same date.
The phylogenetic analyses focused on 16S ribosomal DNA (16S), the most widely sequenced gene fragment for atyid shrimps. From the 16S analyses, we identified a well-supported clade containing the new species and its most closely related taxa. We then constructed a smaller dataset using species from this clade and the second most common fragment sequenced for atyids, the 3' portion of cytochrome c oxidase I (COI). This was undertaken to explore relationships within the closely related species using the faster evolving COI gene.
The 16S dataset included all Caridina species so far reported from Australia and was compiled from the Leichhardt Springs sequences, 12 Australian atyid specimens sequenced for this study, and existing sequences downloaded from GenBank (Table 1). This dataset was aligned using Muscle (Edgar 2004) within Mega version 6 (Tamura et al. 2011). The most appropriate substitution model (lowest Bayesian Information Criterion score) was chosen with Mega for use in the phylogenetic analyses. Two forms of phylogenetic analyses were performed: maximum likelihood analyses in Mega using the appropriate model and bootstrapped 500 times; and Bayesian analyses using MrBayes version 3.2 (Huelsenbeck & Ronquist 2001) with the relevant molecular model for each dataset (parameters: 3 million generations, trees sampled every 1000 cycles, 25% burn in, two runs of four chains heated to 0.2). Genetic distances between species were calculated in Mega using the standard Kimura 2-parameter model (K2P). A smaller 3' COI dataset (species from the well-supported 16S clade containing Caridina biyiga sp. nov.), as well as a combined 16S/3' COI dataset, were also created, and analysed as above.  Wilson. Caridina 'sp. WA4', MAGNT Cr019236, 1 unsexed (3.9 mm CL), Howard River at Gunn Point road crossing, Northern Territory, D. Wilson. Etymology. An indigenous word from the Gundjeihmi language (pronounced bee-yee-ga) meaning 'different' or 'other', a reference to its unusual morphology compared to other species of Caridina. To be used as a noun in apposition.
The large ova are similar in size to the ova of C. formosae Hung, Chan & Yu, 1993 (ova 1.04 × 0.68 mm), a species with highly suppressed, larval development and an almost fully formed, benthic hatchling (Shy et al. 2001).
Colour. Translucent with rust brown chromatophores. Chromatophores either small and widely spaced giving speckled appearance (Fig. 5B) or forming large blotches (Fig. 5A), blotches tending to be grouped as transverse bands on abdomen and posterodorsal carapace, widely spaced on ventral carapace and pereiopods, closely spaced on anterodorsal carapace. Ova rust brown (Fig. 5A).
Habitat. Leichhardt Springs has two dominant outflows approx. 320 m apart-a north arm and a south arm, with a downstream confluence into the northern branch of Burdulba Creek (Fig. 6A). The new species has so far been collected from both the north arm and south arm as far downstream as the junction between the two streams. The total length of watercourse in which the species is presently known is approx. 700 m.
The heat source of Leichhardt Springs is presently undetermined although other thermal springs in the Alligator Rivers Region (including western Arnhem Land) are known to be associated with high heat-producing rocks in the basement of the crust (S. Marshall, Geosciences Australia, pers. comm.). No other thermal springs are presently known from Kakadu NP.
The thermal properties of Leichhardt Springs were investigated over a four to five-month period during the 2014 winter-spring dry season using two CTD-Diver multi-parameter dataloggers deployed on the north arm of Leichhardt Springs, one at the head of the spring and the other 160 m downstream (Figs. 6A-B). A third Diver was also deployed in Radon Springs, a non-thermal spring located approx. 6 km to the northeast on the large, outlying, sandstone formation above Leichhardt Springs (Fig. 1). Over the five-month period, water temperature at the head of the north arm spring was an invariant 30.2°C (SD = 0.005) and 160 m downstream, almost constant and only slightly cooler (mean 29.8°C, range 29.2-30.3°C). By comparison, Radon Springs showed a much higher temperature range (mean 26.0°C, range 22.1-29.3°C), thus 1-8°C cooler over the same period (Fig. 6B). A sampling trip to the south arm of Leichhardt Springs in June 2016 revealed thermal properties similar to the north arm.
Apart from its thermal properties, the location of Leichhardt Springs relative to the Kakadu-Arnhem Land plateau and escarpment complex is also interesting. Other sandstone-formation springs in Kakadu NP arise within small gorges and typically at the immediate base of escarpments. Leichhardt Springs is located approx. 900 m away from the western escarpment of an outlying sandstone formation and at a lower elevation (Fig. 5D).
Satellite imagery of Leichhardt Springs shows extensive rainforest vegetation along both arms associated with the permanent, spring-fed outflows (Fig. 6A). The head of the north arm outflows through a patch of small, abraded and bleached, sandstone cobbles. For at least 300 m downstream, the north arm is characterised by a sequence of pools with clean, quartzite sand substrate interspersed with swift-flowing runs and riffles of gravel and cobble (Fig. 5E). Riparian trees include Melaleuca spp., Carallia brachiata, Pandanus aquaticus, Canarium australianum, Syzygium spp. and the monsoon rainforest-specific species, Nauclea orientalis and Melicope elleryana. The understorey comprises climbing vines (Flagellaria indica and Rhaphidophora australasica), climbing ferns (Stenochlaena palustris), groundstorey ferns (Blechnum orientale) and the flowering shrub, Melastoma sp. The macrophyte, Eleocharis sp., is commonly interspersed amongst runs and riffles of the stream.
The new species is abundant amongst leaf litter and the fibrous roots of riparian trees and ferns along the edges of the stream. No shrimps were found in the clear, faster moving water in the runs and riffles.
The downstream distribution of the shrimps beyond the confluence between the north and south arm is presently unknown as is the dependence of the shrimps on constantly warm, spring waters. Apart from water temperature, water quality of Leichhardt Springs is otherwise typical of surface waters draining the highly leached, sandstone formations of Kakadu NP and western Arnhem Land (Short et al. 2013) being high in clarity, acidic, very soft and low in solutes. Key physicochemical variables at Leichhardt Springs were measured on 14 May 2003 and were as follows: turbidity < 1 NTU; pH 5.2; electrical conductivity 16 μS cm -1 ; Ca and K < 0.1 mg L -1 ; Na 0.9 mg L -1 ; Mg 0.3 mg L -1 ; and SO 4 0.2 mg L -1 .
Distribution. Endemic to Leichhardt Springs, Kakadu NP, Northern Territory, Australia. The known distributional range within the watercourse and associated riparian zones of Leichhardt Springs is 0.05 km 2 .
Molecular results. The five specimens of Caridina biyiga sp. nov. sequenced for 16S produced two very closely related haplotypes of 539 bp. The single individual sequenced for the three remaining genes produced the following sequences: 3' COI (557 bp), 5' COI (629 bp) and 28S (500 bp).
The best-fit models of molecular evolution for the molecular datasets were Hasegawa-Kishino-Yano with a gamma shape parameter and an estimated fraction of invariant sites for the 16S dataset, and Tamura 3-parameter with an estimated fraction of invariant sites for the 3' COI dataset. Bayesian and maximum likelihood trees were produced for both datasets and a Bayesian tree for a small combined 16S/3' COI dataset.
The most closely related clade to the 'thermophila' group may be a moderately supported grouping of species all from the Kimberley, WA (Bayesian posterior probability 0.90; not supported by the maximum likelihood analysis). Within this clade, a grouping comprising C. spelunca Choy, 1996;C. 'sp. WA2';C. 'sp. WA5';and C. 'sp. WA6' was moderately supported by both the Bayesian and maximum likelihood analyses (Bayesian posterior probability 0.66; maximum likelihood bootstrap 74%) and is hereby referred to as the 'spelunca' group. K2P genetic distances between 16S sequences of C. biyiga sp. nov. and species from the 'spelunca' group ranged from 7.7-9.0% with a 3' COI distance of 15.9% to C. 'sp. WA2' from that group.
Relationships of the remaining Australian Caridina species were partially resolved using the 16S dataset. Two species groups, which were also easily recognised using morphological characters, were recovered as strongly supported clades in both the Bayesian and maximum likelihood analyses; viz. a 'gracilirostris' group (Bayesian posterior probability 1.00; maximum likelihood bootstrap 99%) and a 'serratirostris' group (Bayesian posterior probability 1.00; maximum likelihood bootstrap 95%).
The Caridina zebra species complex (sensu Choy et al. 2019) also formed a strongly supported clade in both the Bayesian and maximum likelihood analyses (Bayesian posterior probability 0.99; maximum likelihood bootstrap 86%). This complex comprises three, highly similar species from the Tablelands and Cardwell Range areas of the wet tropics, viz. C. zebra Short, 1993, C. confusa Choy & Marshall, 1997  Another strongly supported clade included the species C. 'sp. Gulf2' from northwest Qld and C. 'sp. NT2' from the Darwin area, Northern Territory (Bayesian posterior probability 1.0; maximum likelihood bootstrap 100%), although the comparative morphology of the two species was not investigated in the present study.
Two representatives of the Caridina 'sp. D' complex, C. 'sp. D' and C. 'sp. DG', were recovered as a well-supported clade (1.00 Bayesian posterior probability; 100% maximum likelihood bootstrap). This species complex, which requires further study to resolve relationships between putative species, shows an extensive distribution across much of northern, eastern and central Australia. Material from east Queensland has previously been reported as C. nilotica aruensis (J. Roux 1926;Riek 1953;Glaister 1976) although the genetic relationship between Australian material and C. aruensis J. Roux, 1911 from the Aru Islands, Indonesia, is presently undetermined.  Within the two hypogeal-epigeal species clades reported above, strong relationships between P. gracilis and Pycneus morsitans (Bayesian posterior probability 1.0; maximum likelihood bootstrap 87%) and P. unguis with Pycnisia raptor (Bayesian posterior probability 1.0; maximum likelihood bootstrap 100%) were also recorded. These relationships have previously been reported in the 16S analyses of Page et al. (2008b) andvon Rintelen et al. (2012). The close relationship of P. gracilis and Pycneus morsitans was also confirmed in the combined 16S/28S/H3 and 16S/28S analyses of von Rintelen et al. (2012). The molecular data strongly suggest that Australian Parisia are non-monophyletic and that the current generic classification of Australian species of Parisia, Pycneus and Pycnisia requires re-appraisal.
Systematic Position. Although the general morphology of the new species is typical of Caridina, the branchial formula, fingertips of the chelae and fifth pereiopod dactylus are unusual for the genus.
The new species lacks an arthrobranch at the base of the first cheliped and has 8 pairs of branchiae rather than the full complement of 9 branchiae normally associated with Caridina. There is also a vestigial epipod on the first maxilliped ( Fig. 3F) in addition to the epipods on maxillipeds 2-3 (Figs. 3D, G) and pereiopods 1-4 ( Fig. 4J) typical of Caridina. Under the subfamilial and generic classification of Holthuis (1993), these differences would exclude the new species from Caridina and would assign the species to a different subfamily (Caridellinae rather than Atyinae). However, recent studies (von Rintelen et al. 2008;von Rintelen et al. 2012;De Grave & Page 2014) indicate that the branchial formula is less conservative within the Atyidae than previously accepted and minor differences in the development of the branchiae are not reliable for defining genera and subfamilies. For example, Caridinides wilkinsi Calman, which was recently transferred to Caridina by De Grave & Page (2014), has an exopod on the first pereiopod and a small epipod on the first maxilliped. Similarly,  noted that the arthrobranch at the base of the first cheliped is sometimes reduced in C. serratirostris, and invariably absent in C. celebensis De Man, 1892. In C. thomasi von Rintelen, Karge & Klotz, 2008, there is only one arthrobranch rather than the usual two arthrobranchs at the base of the third maxillipeds. The branchial formula for C. biyiga sp. nov. falls within the wide range of variation now reported for Caridina sensu lato.
The fifth pereiopod dactylus of the new species is also atypical for the genus and is similar in shape to the dactyli of the third and fourth pereiopods K). In other species of Caridina, the fifth pereiopod dactylus is usually more slender and elongate than the preceding ambulatory pereiopods, as illustrated by Figs. 13F-I, 14E-H, 15G-J) for the type species, C. typus. In the new species, the number of spiniform setae on the flexor margin of the dactylus is also similar to the preceding two pereiopods. At most, there are four, widely spaced, claw-like spiniform setae on the flexor margin (Fig. 4K). In other Caridina species, including C. typus (see Figs. 13I, 14H, 15J), there is a comb-like row of numerous, closely spaced setae.
In the new species, the fingers of the first and second chelae are distinctive for the genus in having flexible, spatulate tips (Fig. 2H). In the type species of the genus, C. typus, the fingertips bear a rigid, hooked, nail-like unguis as illustrated by Bouvier (1925;Fig. 282). Within the described Australian Caridina fauna, the new species is easily recognised by the short, slender, depressed rostrum bearing 3-7 longitudinally compressed, preorbital, dorsal teeth but lacking ventral teeth (Figs. 2A,E,; the anteromedially produced scaphocerite lamina (Fig. 2F); and the relatively short, first and second chelae which have length/breadth ratios less than 1. .
In regard to the short, slender, depressed rostrum, the new species shows some resemblance to C. confusa, C. malanda, C. spinula, and C. zebra from northeast Qld, but differs in having 3 or more dorsal teeth (Figs. 2A, E, I-J). It also differs in having the scaphocerite lamina anteromedially produced vs. anteromesially produced; 4 or fewer spiniform setae on the flexor margin of P5 dactylus vs. 45 or more spiniform setae on the flexor margin of P5 and in lacking an arthrobranch at the base of the first chelipeds.
Although the 16S molecular analyses placed the new species in a strongly supported clade, the 'thermophila' species group, these relationships are not readily apparent using morphological characters. In addition to the characters mentioned above for distinguishing the new species from its Australian congeners in general, the new species can easily be distinguished from C. thermophila and C. 'sp. NT1' within the 'thermophila' group, by the following characters: 1. rostrum depressed, with 7 or fewer dorsal teeth and lacking postorbital teeth ( Figs Recent COI meta-analyses for the Decapoda are helpful to some degree regarding genetic evidence for or against the recognition of a new genus to accommodate the new species. The closest sister taxon to the new species based on the 3' COI fragment was C. 'sp. NT1' in the 'thermophila' species group with a K2P distance of 10.4%. This is slightly below the 11.27-49.93% range of genetic difference between genera within a family reported by Costa et al. (2007) and significantly lower than the 19.75% average they reported. It is also lower than the 22.33% average reported by Matzen da Silva et al. (2011) for decapods but falls within the large overall range between genera of 6.69-48. 35%.
Although both the genetic and morphological data indicate that C. biyiga sp. nov. is a highly distinctive species within the Australian Caridina fauna, there does not appear to be sufficient evidence at this point to warrant the recognition of a new monotypic genus. The genetic analyses placed the new species in the 'thermophila' group, a strongly supported clade including at least one typical species of Caridina, viz. C. thermophila. The K2P genetic distances between the new species and other species within the clade are also towards the lower end of the range recorded between genera in decapod meta-analyses (Costa et al. 2007;Matzen da Silva et al. 2011). We therefore assign the new species to the genus Caridina.
The following illustrated key can be used to identify the new species from other Northern Territory Caridina. The inclusion of C. typus in the key is based solely on the Australian distribution given by Horwitz (1995), 'Northeastern Queensland to Northern Territory', which is possibly erroneous. Although the species is known from eastern Queensland, we have so far been unable to find other records or material of the species from the Northern Territory.
Among the endemic Northern Territory Caridina, C. biyiga sp. nov. shows by far the most restricted distribution and specialised habitat requirements. The new species also shows 16S genetic distances from other species in the 'thermophila' group of 5.1-6.0%. This roughly equates to a late Miocene origin, based on a commonly used divergence rate for decapod crustaceans of 0.65-0.9% per million years (Schubart et al. 2000). In addition to its distinctive genetics, the new species also displays highly distinctive morphology compared to its congeners.
To further highlight the distinctive fauna and conservation significance of Leichhardt Springs, two freshwater fishes with distinctive genetics have also been collected from the same locality: a highly divergent, infraspecific lineage of the Spotted Blue Eye, Pseudomugil gertrudae (Cook et al. 2014) and a cryptic species of rainbowfish related to Melanotaenia trifasciata (Unmack et al. 2013). A current phylogenetic study of the phreatoicidean isopod families Amphisopidae and Mesamphisopidae (Wilson and Humphrey in prep.) has also revealed a new genus and species from Leichhardt Springs. The species was previously reported in Wilson et al. (2009) as 'sp. 21' among a large flock of undescribed Eophreatoicus species (>30 species) from the Alligator Rivers Region.