Two new species of Nitocrella (Crustacea, Copepoda, Harpacticoida) from groundwaters of northwestern Australia expand the geographic range of the genus in a global hotspot of subterranean biodiversity

In Australia, the Ameiridae is the most diverse harpacticoid family in groundwater, with 35 species hitherto reported. In this study, we describe two new species belonging to the “vasconica”-group of the ameirid genus Nitocrella based on specimens collected from groundwaters near mine sites in the Pilbara and Great Sandy Desert regions of northwestern Australia. Nitocrella knotti sp. n. can be distinguished from related taxa by having two setae on the antennal exopod, four armature elements on the distal endopodal segment of leg 2, four armature elements on the distal endopodal segment of leg 3, three armature elements on the distal endopodal segment of leg 4, and three setae on the basoendopodal lobe of leg 5. Nitocrella karanovici sp. n. differs from its congeners by having a short outer spine and long inner seta on the distal endopodal segment of leg 2, three armature elements on the distal endopodal segment of leg 3, and four setae on the basoendopodal lobe of leg 5 in the female. This study is of biogeographic interest in providing the first documentation of the genus Nitocrella from the Pilbara and Great Sandy Desert regions. Both new species of Nitocrella are recorded from restricted localities and appear to be short-range endemics, thus making them potentially vulnerable to environmental changes and threatening processes such as mining. The distribution range of N. karanovici sp. n. coincides with the centre of diversity of the Ethel Gorge aquifer stygobiont community, a globally significant hotspot which is listed as endangered. Subterranean Biology 20: 51–76 (2016) doi: 10.3897/subtbiol.20.10389 http://subtbiol.pensoft.net Copyright Danny Tang, Stefan M. Eberhard. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal


Introduction
The family Ameiridae (Copepoda) has successfully colonized and radiated in continental surface water and groundwater, with over 150 species reported from Australia, Asia, Europe, and North America Halsey 2004, Boxshall andDefaye 2008). In Australia, the Ameiridae is the most diverse harpacticoid family in groundwater (Karanovic 2006, Karanovic andHancock 2009). Presently 35 ameirid species have been reported from groundwater, mostly in Western Australia and a few in Queensland and South Australia ( Table 1).
Groundwaters of (semi-) arid Western Australia are a globally significant hotspot for subterranean biodiversity (Humphreys 2008, Eberhard et al. 2009, Guzik et al. 2011). Most of this rich diversity occurs in two adjacent geographic regions, the Pilbara and the northern Yilgarn, both of which form parts of the Western Shield, a single emergent land mass since the Proterozoic (Fig. 1A). In both regions, progressive Quaternary climatic aridity is considered the major driver for groundwater colonization; however, there are some remarkable differences between the copepod and other stygofauna taxa of the Pilbara and northern Yilgarn, which have almost no genera in common and exhibit major differences in higher taxa (Karanovic 2006, Humphreys 2008, 2012). An explanation for this great biogeographic disjunction remains elusive.
The majority of ameirid species known from groundwater in Australia were collected at, or adjacent to, proposed mine sites which have been surveyed for stygofauna as part of the mine project environmental impact assessment, or, as part of the ongoing environmental compliance monitoring at established mine sites. Other ameirids were collected from pastoral wells and groundwater boreholes during the course of surveys by government departments, museums, and universities. Most of the described ameirids are recorded from single localities and appear to be short-range endemic species (sensu Harvey 2002, and this makes them potentially vulnerable to environmental changes and threatening processes such as mining and groundwater abstraction. In some cases this has led to conflict between the competing interests of biodiversity conservation and resources development (see Karanovic et al. 2013). In this study, we describe two new ameirid species belonging to the genus Nitocrella Chappuis, 1923 based on specimens collected from groundwaters near two mine sites in the Pilbara and Great Sandy Desert regions of northwestern Western Australia.

Methods
The net-haul method (see Eberhard et al. 2005Eberhard et al. , 2009) was used to collect samples of stygofauna in July 2008 from one borehole located 48 km from the Telfer Mine in Table 1. Harpacticoid copepods of the family Ameiridae reported from subterranean waters of Australia (in alphabetical order).

Order
Description. Female. Body ( Fig. 2A) subcylindrical, 610-635 µm (mean 620 µm; n = 3) long (measured from tip of rostrum to posterior margin of caudal rami) and 148-155 µm (mean 153 µm; n = 3) wide (at posterolateral margin of cephalothorax). Prosome composed of cephalothorax and 3 free pedigerous somites; tergite of first two pedigerous somites each with elliptical integumental window. Urosome comprised of fifth pedigerous somite, genital double-somite, and 3 free abdominal somites. Fifth pedigerous somite with short, dorsolateral row of spinules and numerous short rows of minute denticles (not drawn) and minute surface pits (not drawn) on dorsal and ventral surfaces. Components of genital double-somite ( Fig. 2A, B) partially fused dorsally but completely fused ventrally, ornamented with short, anterolateral row of spinules on ventral surface, minute surface pits and numerous short rows of minute denticles on dorsal and ventral surfaces (only minute denticles on ventral surface are shown), and row of large spinules and serrated hyaline frill encircling posterior margin; genital field with large median copulatory pore, chitinized copulatory duct leading anteriorly to pair of bilobate seminal receptacles, and median genital pore covered by operculiform leg 6. First free abdominal somite with large integumental window on ventral surface, minute surface pits and numerous short rows of minute denticles on dorsal and ventral surfaces (only minute denticles on ventral surface are shown), and row of large spinules and serrated hyaline frill ringing posterior margin. Second free abdominal somite similar to preceding somite, but without integumental window. Anal somite (Fig. 2B, C) with minute surface pits (not drawn) and numerous short rows of minute denticles on dorsal and ventral surfaces and row of large spinules along posterior border and along posterior margin of anal operculum.
Legs 1-4 biramous (Figs 3F, G, 4A, B); leg 1 with trimerous rami; legs 2-4 with trimerous exopod and bimerous endopod. Armature on rami of legs 1 to 4 as follows (Roman numerals = spines; Arabic numerals = setae): Coxa with 2 rows of minute spinules and 1 row of large spinules on anterior surface; outer margin with 1 row of large spinules and 2 rows of fine spinules; inner distal corner with row of fine spinules. Basis with row of large spinules at insertion of each ramus and row of fine spinules along inner margin and on posterior surface; 1 additional large spinule present near base of inner spine; both spines with subapical flagellum. Outer spine on proximal exopodal segment with subapical flagellum. First two exopodal segments with large spinules along outer margin and on outer distal corner, as well as fine spinules along inner margin; distal segment with large spinules along outer margin and 1 spinule on apical margin. Both setae on terminal exopodal segment geniculate. Proximal endopodal segment long, extending almost to mid-point of distal exopodal segment, with fine spinules along inner margin; middle segment with fine spinules along outer and inner margins; distal segment with fine spinules along outer margin. Two (of 3) setae on distal endopodal segment geniculate. Both rami with minute surface pits (not drawn).
Leg 2 (Fig. 3G) intercoxal sclerite posteriorly bilobate, with row of spinules on each lobe. Coxa with 4 rows of minute spinules on anterior surface; outer margin with 2 rows of fine spinules; inner distal corner with row of minute spinules. Basis ornamented as in leg 1, except lacks large spinule near inner margin and with additional row of minute spinules on anterior surface. Exopod ornamented as in leg 1, except with additional spinulated frill on inner distal corner of proximal and middle segments. First two exopodal segments protruded on outer distal corner. Proximal endopodal segment with spinules along outer and inner margins and short spinulated frill on inner distal corner. Distal endopodal segment about 1.5 times longer than proximal endopodal segment and furnished with spinules along outer and inner margins. Both rami with minute surface pits (not drawn) as in leg 1.
Leg 3 (Fig. 4A) similar to leg 2, except with naked intercoxal sclerite, outer seta (instead of spine) on basis, longer inner spine on middle exopodal segment, and inner distal seta (instead of spine) and longer inner proximal spine on distal endopodal segment.
Leg 4 (Fig. 4B) similar to leg 3, except with smaller intercoxal sclerite, row of spinules absent on posterior surface of basis, less protruded outer distal corner on first two exopodal segments, shorter inner spine on middle exopodal segment, 2 more elements on distal exopodal segment, shorter distal endopodal segment, and only 3 elements on distal endopodal segment.
Leg 6 ( Fig. 2B) represented by simple operculum covering genital pore, armed with 1 minute naked seta on outer distal corners.
Etymology. This species is named in honour of the late Professor Brenton Knott (The University of Western Australia) who made significant contributions to research on groundwater fauna in Western Australia.
Differential diagnosis. Among the three groups of Nitocrella proposed by Petkovski (1976), i.e. "chappuisi", "hirta", and "vasconica", Nitocrella knotti sp. n. belongs to the "vasconica"-group as it also possesses the characteristic six armature elements on the distal exopodal segment of leg 4. With the addition of N. knotti sp. n. (including the second new species described below), this group currently contains 21 species reported from Eurasia, the Caribbean, and Australia (Table 2). Nitocrella knotti sp. n. shares with N. afghanica Štĕrba, 1973, N. jankowskajae Borutzky, 1972, N. kirgizica Borutzky, 1972, N. monchenkoi Borutzky, 1972, N. obesa Karanovic, 2004, and N. trajani Karanovic, 2004 an armature formula of I-0; I-I; II,2,0 on the exopod and 0-I; 0-0; 0,3,0 on the endopod of leg 1, II,2,0 on the distal exopodal segment of legs 2 and 3, and 0-I on the proximal endopodal segment of legs 2-4. However, N. knotti sp. n. can be easily distinguished from those taxa by having four armature elements (instead of two for N. jankowskaja, or three for N. afghanica, N. kirgizica, N. monchenkoi, N. obesa, and N. trajani) on the distal endopodal segment of leg 2. Nitocrella knotti sp. n. differs further from the Australian N. obesa and N. trajani by having the genital and first abdominal somites fused ventrally (rather than completely separate), two setae (rather than three) on the antennal exopod, and three setae (rather than four) on the basoendopodal lobe of leg 5, among others; and from the Central Asian N. afghanica, N. jankowskajae, N. kirgizica, and N. monchenkoi by having four armature elements (instead of two) on the distal endopodal segment of leg 3 and three armature elements (instead of one for N. afghanica, or two for N. jankowskaja, N. kirgizica, and N. monchenkoi) on the distal endopodal segment of leg 4. Table 2. Species of Nitocrella belonging to the "vasconica"-group (in alphabetical order).
Other material examined. All material collected from boreholes in the Ethel Gorge aquifer, approximately 15 km ENE of Newman, Western Australia (Fig. 1) Description. Female. Body (Fig. 5A) cylindrical, 450-495 µm (mean 471 µm; n = 6) long (measured from tip of rostrum to posterior margin of caudal rami) and 95-105 µm (mean 103 µm; n = 6) wide (at first free pedigerous somite). Prosome composed of cephalothorax and 3 free pedigerous somites. Urosome comprised of fifth pedigerous somite, genital double-somite, and 3 free abdominal somites. Components of genital double-somite ( Fig. 5A, B, C) not fused dorsally but completely fused ventrally, with elliptical integumental window laterally, row of small spinules immediately posterior to each integumental window, and row of large spinules and frill of minute spinules encircling posterior margin; genital field with large median copulatory pore, chitinized copulatory duct leading anteriorly to pair of lobate seminal receptacles, and median genital pore covered by operculiform leg 6. First free abdominal somite with anteroventral pair of oval integumental windows and row of unequal spinules and frill of minute spinules ringing posterior border. Second free abdominal somite with row of subequal spinules and frill of minute spinules encircling posterior edge. Anal somite (Fig. 5B, D, E) with anterior and posterior row of spinules on ventral surface, several rows of spinules on lateral surface, and spinules along posterior margin of anal operculum.

Coxa Basis Exopod Endopod
Leg 1  Leg 1 (Fig. 7A) intercoxal sclerite naked and concave on posterior margin. Coxa with 1 row of spinules on anterior surface and another row of spinules on posterolateral surface. Basis with row of long spinules at insertion of each ramus and 3 additional large spinules proximal to inner spine; both spines with subapical flagellum. Exopodal segments with large spinules along outer margin and on outer distal corner; middle segment also with fine spinules along inner margin. Endopodal segments with large spinules on outer margin and fine spinules along inner margin. Both setae on terminal exopodal segment and 1 of 3 setae on distal endopodal segment geniculate.
Leg 2 (Fig. 7B) intercoxal sclerite naked and posteriorly bilobate. Coxa with 1 row of minute spinules on posterolateral surface. Basis with row of spinules at insertion of each ramus and several fine spinules (only 1 depicted) on inner margin; outer spine with subapical flagellum. Exopod ornamented as in leg 1, except with additional spinulated frill on inner distal corner of proximal and middle segments. First two exopodal segments protruded on outer distal corner. Both endopodal segments with spinules along outer margin.
Leg 3 (Fig. 7C) similar to leg 2, except with outer seta (instead of spine) on basis and 3 elements on distal endopodal segment.
Leg 4 (Fig. 7D) similar to leg 3, except with much smaller spinules at insertion of endopod, 6 elements on distal exopodal segment (of which inner distal seta is longer and ornamented with tightly packed spinules on inner margin of apex), and 2 elements on distal endopodal segment.
Leg 6 ( Fig. 5B) represented by genital operculum covering genital pore, and armed with 1 minute naked seta on distolateral borders.
Inner spine on basis of leg 1 (Fig. 8C) modified as is typical for members of Ameiridae.
Leg 6 ( Fig. 8A) asymmetrical, with right side modified as operculum and left side basally fused to somite; each side armed with 2 unequal distolateral setae.
Variability. One paratype female with discontinuous row of spinules along posteroventral margin of anal somite (Fig. 5B), but row is continuous in other paratype specimens. One dissected paratype female and 1 intact paratype male with 4 elements on terminal exopodal segment of leg 1 (Fig. 8E). One dissected and 1 intact paratype males with 3 elements on distal endopodal segment of leg 2 (Fig.  9C). One dissected paratype female with 4 elements on terminal exopodal segment of leg 3 (Fig. 8F). One dissected paratype female and 1 intact paratype male with 2 elements on distal endopodal segment of leg 3 (Fig. 8G). One dissected and 3 intact paratype females with longer inner distal spine on distal endopodal segment of leg 3 (Fig. 8H). One dissected and 3 intact paratype females plus 3 intact paratype males with 3 elements on distal endopodal segment of leg 4 (Fig. 8I). One intact paratype female with 1 element on distal endopodal segment of leg 4 (Fig. 8J). Two dissected and 1 intact paratype females with 3 ( Fig. 9A) or 2 elements (Fig. 9B) on basoendopod of leg 5. Five intact paratype males with 1 (Fig. 9D) or no elements (not drawn) on basoendopod of leg 5. One intact paratype male with 3 setae on leg 6 (not drawn).
Etymology. This species is named for Dr. Tomislav Karanovic, in recognition of his extensive taxonomic research on subterranean copepods of Australia.
Differential diagnosis. Nitocrella karanovici sp. n. also belongs to the "vasconica"group as it possesses the distinctive six armature elements on the distal exopodal segment of leg 4. Of the other 20 species in this group, N. karanovici sp. n. shares five armature elements on the distal exopodal segment of leg 1 with only N. dussarti Rouch, 1959 andN. gracilis Chappuis, 1955. Nitocrella karanovici sp. n. can be easily distinguished from N. dussarti by having three armature elements (instead of four) on the distal endopodal segment of leg 3 and four setae (instead of three) on both the exopod and basoendopodal lobe of leg 5 in the female, and from N. gracilis by having a short outer spine and long inner seta (rather than two subequal setae) on the distal endopodal segment of leg 2, two spines and one seta (instead of one spine and two setae) on the distal endopodal segment of leg 3, and four setae (instead of 3) on the basoendopodal lobe of leg 5 in the female.

Discussion
This study is of biogeographic interest in providing the first documentation of the genus Nitocrella from the Pilbara and Great Sandy Desert regions of northwestern Australia. Previously in Australia, Nitocrella was known only from three species in the northern Yilgarn region (Karanovic 2004) (Fig. 1A). Karanovic (2006) recognized that the subterranean copepod fauna is strikingly dissimilar, particularly at the genus level, between the neighboring Pilbara and Yilgarn regions (of which the Murchison region forms a part). Prior to this study, only Schizopera G. O. Sars, 1905 and Nitocrellopsis Galassi, De Laurentiis & Dole-Olivier, 1999 were known from both regions. Indeed, the Pilbara region has stronger copepod faunal connections to the Kimberley region in Western Australia, and to northern Queensland, than to the northern Yilgarn region (Karanovic 2006, Karanovic andHancock 2009). This study brings the number of copepod genera now known to be shared between the Pilbara and northern Yilgarn to three, although the addition of this third copepod genus makes little difference to further understanding of the perplexing taxonomic dissimilarities between these two adjacent stygo-regions. Karanovic (2010) noted that the species of Nitocrellopsis reported from the two regions are only remotely related to each other. The same trend also appears to be the case for the species of Nitocrella. For example, although all five species of Nitocrella collected from Western Australia belong to the "vasconica"-group, they differ from each other in many respects (see Table 3). Nitocrella karanovici sp. n. and N. knotti sp. n. both have the genital and first abdominal somites fused ventrally, but they differ in the number and position of the integumental windows, the armature of the antennal exopod, and the armature of legs 1 to 5. Nitocrella obesa and N. trajani share the same number of armature elements on legs 1, 2 and 4, but they differ in the body ornamentation and armature of legs 3 and 5. Nitocrella absentia Karanovic, 2004 and N. karanovici sp. n. have the same armature on the exopod of legs 2 and 3 and on the basoendopod of leg 5, but they differ in the number and ornamentation of the urosomites and armature of leg 4. The collection records for both Nitocrella karanovici sp. n. and N. knotti sp. n. strongly suggest that each species is a short-range endemic (SRE), with their recorded distribution ranges being much less than the SRE thresholds of 10 000 km 2 nominated by Harvey (2002), or 1 000 km 2 recommended by Eberhard et al. (2009) for Pilbara stygofauna. Both collection localities have been subjected to multiple monitoring surveys over some 15 years. Out of 30 boreholes monitored in eight surveys at Telfer, Nitocrella sp. has only been recorded from two closely adjacent boreholes (HB405 and HB406) (Bennelongia 2010). Other stygofaunal taxa collected from borehole HB405 included copepods, ostracods, and paramelitid amphipods. The aquifer type, groundwater depth, and physicochemistry in the Telfer boreholes were not available to this study.
Eighty-four species of stygofauna have been recorded from the Ethel Gorge aquifer and adjacent groundwaters in the Newman area, thus ranking it as one of the richest localized groundwater fauna assemblages in Australia, and indeed globally (Subterranean Ecology 2013, 2014, Halse et al. 2014). At least 45 species, including N. karanovici sp. n., are considered to be stygobionts (obligate groundwater species) because they possess morphological specializations to subterranean life and are not known from surface waters. Around 40 of the stygobiont species have to date only been recorded from the Ethel Gorge aquifer or adjacent groundwaters in the Newman area (Subterranean Ecology 2013). The Ethel Gorge aquifer stygobiont community is therefore a local hotspot within a regional hotspot. The centre of species richness and abundance for this stygobiont community is concentrated in the shallow alluvial and calcrete aquifer around Ethel Gorge where the Fortescue River flows through the Ophthalmia Range. It is effectively a subsurface gorge where the bedrock shallows and the watertable lies generally less than ten metres below ground level (BHP undated). The distribution range of N. karanovici sp. n. more or less coincides with this core centre of stygobiont community richness which extends upstream of the gorge for approximately 2 km and downstream for approximately 6 km (Bennelongia 2015) (Fig. 1B). The groundwater quality in the shallow aquifer is predominantly fresh with measured salinities in most boreholes less than 1500 mg/L but with some boreholes recording salinities up to around 5,000 mg/L (Subterranean Ecology 2014). The highest salinity groundwater that N. karanovici sp. n. was collected from was around 2400 mg/L. The Ethel Gorge aquifer stygobiont community is listed in Western Australia as an endangered Threatened Ecological Community (TEC) by the Department of Parks and Wildlife (DPaW 2014). The Environmental Protection Authority (EPA 2016) has identified potential impacts on stygofauna habitat and species within the Ethel Gorge TEC from mine dewatering groundwater drawdown and changes in water quality due to the discharge of surplus water into Ophthalmia Dam. As a consequence of its proximity to these potential threatening processes the Ethel Gorge aquifer has received greater sustained survey effort (mostly in the form of environmental compliance monitoring) and taxonomic scrutiny (including molecular genetic studies) than any other groundwater system in Australia. For environmental impact assessment (EIA) and environmental compliance monitoring it is pertinent to note that even considering the intensive biannual field sampling and specimen identification efforts over more than 15 years, new species (including N. karanovici sp. n.) continue to be detected from boreholes that have been sampled many times previously (Subterranean Ecology 2012. While this finding is entirely consistent with intensively studied groundwater systems elsewhere in the world (e.g. Culver and Pipan 2009, Dole-Olivier et al. 2015, Brancelj et al. 2016, this facet of subterranean biology is not widely recognised in EIA for mine projects in Australia.