Taking eDNA underground: Factors affecting eDNA detection of subterranean fauna in groundwater.

Stygofauna are aquatic fauna that have evolved to live underground. The impacts of anthropogenic climate change, extraction and pollution on groundwater pose major threats to groundwater health, prompting the need for efficient and reliable means to detect and monitor stygofaunal communities. Conventional survey techniques for these species rely on morphological identification and can be biased, labour-intensive and often indeterminate to lower taxonomic levels. By contrast, environmental DNA (eDNA)-based methods have the potential to dramatically improve on existing stygofaunal survey methods in a large range of habitats and for all life stages, reducing the need for the destructive manual collection of often critically endangered species or for specialized taxonomic expertise. We compared eDNA and haul-net samples collected in 2020 and 2021 from 19 groundwater bores and a cave on Barrow Island, northwest Western Australia, and assessed how sampling factors influenced the quality of eDNA detection of stygofauna. The two detection methods were complementary; eDNA metabarcoding was able to detect soft-bodied taxa and fish often missed by nets, but only detected seven of the nine stygofaunal crustacean orders identified from haul-net specimens. Our results also indicated that eDNA metabarcoding could detect 54%-100% of stygofauna from shallow-water samples and 82%-90% from sediment samples. However, there was significant variation in stygofaunal diversity between sample years and sampling types. The findings of this study demonstrate that haul-net sampling has a tendency to underestimate stygofaunal diversity and that eDNA metabarcoding of groundwater can substantially improve the efficiency of stygofaunal surveys.

Macedomeris, a new monotypic doderiinine genus (Diplopoda, Glomerida) from a cave in North Macedonia, Balkan Peninsula.

Here we describe a new monotypic glomerid genus, Macedomeris gen. nov., with Macedomeris ivoi sp. nov. (a presumed troglobiont) as its type species. The new genus clearly differs from all other genera within the order Glomerida by the combination of several morphological characters: the presence of a characteristic deep lateral pit ("Ohrgrube") on both sides of the thoracic shield, the absence of striking ornamentation on tergites, the fusion of tergite 11 with the anal shield, and the lack of a large medial hump. In addition, Macedomeris gen. nov. differs from other members of the Glomerida by its general appearance, the presence of 2 or 3 vestigial ommatidia and a very wide syncoxite of a 2-segmented leg-pair 17 in the male. Notes on its ecology, troglomorphic features, and relationships with similar members of the tribe Doderiini are also given.

Scratching the surface of subterranean biodiversity: Molecular analysis reveals a diverse and previously unknown fauna of Parabathynellidae (Crustacea: Bathynellacea) from the Pilbara, Western Australia.

Like other crustacean families, the Parabathynellidae is a poorly studied subterranean and aquatic (stygobiontic) group in Australia, with many regions of available habitat having not yet been surveyed. Here we used a combined approach of molecular species delimitation methods, applied to mitochondrial and nuclear genetic data, to identify putative new species from material obtained from remote subterranean habitats in the Pilbara region of Western Australia. Based on collections from these new localities, we delineated a minimum of eight and up to 24 putative new species using a consensus from a range of molecular delineation methods and additional evidence. When we placed our new putative species into the broader phylogenetic framework of Australian Parabathynellidae, they grouped with two known genera and also within one new and distinct Pilbara-only clade. These new species significantly expand the known diversity of Parabathynellidae in that they represent a 22% increase to the 109 currently recognised species globally. Our investigations showed that sampling at new localities can yield extraordinary levels of new species diversity, with the majority of species showing likely restricted endemic geographical ranges. These findings represent only a small sample from a region comprising less than 2.5% of the Australian continent.

The age and diversification of metacrangonyctid subterranean amphipod crustaceans revisited.

The Metacrangonyctidae are a small family of amphipod crustaceans of marine origin found only in subterranean continental waters. They display a broad but punctuated distribution between the Caribbean and the Arabian Peninsula, with major disjunctions either due to vicariance by plate tectonics or to occurrence of recent episodes of long-distance transoceanic dispersal. We re-examine the phylogeny of the family and the time frame for its diversification using mitochondrial genomes in the light of two key taxa recently discovered, from Oman (Arabian Peninsula) and the Rif area of Morocco, respectively. We also use a novel fossil calibration scheme of the mitogenome phylogeny. Results of previous analyses based on palaeogeographic calibrations are not contradicted by the new approach, with vicariance by plate tectonics remaining as the main explanatory factor for the amphi-Atlantic distribution displayed by this ancient group of subterranean amphipods.

Coming out in a harsh environment: a new genus and species for a land flatworm (Platyhelminthes: Tricladida) occurring in a ferruginous cave from the Brazilian savanna.

Faunal inventories in ferruginous caves from an area belonging to the Brazilian savanna (Cerrado phytophysiognomy), on the eastern margin of the Serra do Espinhaço Plateau, in southeastern Brazil, have revealed the occurrence of land flatworms. Herein, a flatworm sampled in such subterranean environment is described as a new genus and species of the Neotropical subfamily Geoplaninae, Difroehlichia elenae gen. nov., sp. nov. The new genus shows rare features within Geoplaninae, namely sub-cylindrical body, poorly developed sub-epidermal musculature and a narrow creeping sole. Some features, such as a small body and a broad sensory margin in the anterior region of the body, as well as the fact that the holotype showed signs of recent copula, may indicate an adaptation to the subterranean environment, probably representing a troglophile. Difroehlichia elenae is characterized by an almost homogeneous dark brown pigmentation over dorsal surface and body margins, a short cylindrical pharynx, and a tubular and unforked intrabulbar prostatic vesicle, among other features. The holotype shows a secondary male copulatory organ located immediately behind the primary one, both communicating with the female atrium and gonopore canal. Since the species seems to have low abundance and restricted distribution and its type-locality is affected by mining activities, major concern arises regarding its conservation.

Reassessment of the Groundwater Amphipod Paramoera relicta Synonymizes the Genus Relictomoera with Paramoera (Crustacea: Amphipoda: Pontogeneiidae).

The systematic status of the stygobitic amphipod, Paramoera relicta Uéno, 1971, was revisited based on morphological examination of the paratypes, and molecular phylogenetic analyses using a newly collected specimen from its type locality. The paratypes clearly showed that this species does not possess the "sinusoid" antennal sinus in the head, which was erroneously described in the original description of this species; P. relicta is unquestionably classified within the genus Paramoera Miers, 1875 . Molecular phylogenetic trees obtained with nuclear histone H3 and mitochondrial 16S rRNA demonstrated that P. relicta clearly belongs to the clade consisting of Paramoera species. Since the genus Relictomoera Barnard and Karaman, 1982 was erected according to the "sinusoid" characteristic of its type species P. relicta, this genus is no longer valid, and should be treated as a junior subjective synonym of Paramoera. One of the paratypes of P. relicta is herein designated as a neotype for the species to clarify its taxonomic status and emend the morphological characteristics of this pontogeneiid amphipod.

Discovery of representatives of the family Nicoletiidae (Insecta: Zygentoma) from caves of Iran, with descriptions of two new supraspecific taxa.

The first records of the family Nicoletiidae in Iran are given, based on material from subterranean habitats in Fars Province. Two new species are described, one belonging to a new genus of Atelurinae, Persiatelurina farsiana gen. n. sp. n., the other to the genus Lepidospora but representing a new subgenus, Brinckiletinia subgen. n., this new subgenus is close to the subgenus Brinckina, but lacks scales on the thorax. Both new taxa are compared with related species. Nipponatelurina caucasica Kaplin, 2016 is transferred to Persiatelurina caucasica n. comb. The new atelurine was collected in Momtaz and Khan Caves and Lepidospora (Brinckiletinia) malousjanica sp. n. was found in Malousjan Cave. An additional specimen of Lepidospora was collected in Momtaz Cave, and although its specific identity is not clear, it represents a third taxon belonging to the subgenus Brinckina. Data about the habitats of these subterranean Nicoletiidae are presented.

A reassessment of the phylogenetic utility of genus-level morphological characters in the family Bogidiellidae (Crustacea, Amphipoda), with description of a new species of Eobogidiella Karaman, 1981.

Bogidiellidae is the most diverse and cosmopolitan family of stygobiotic amphipods, and inhabits a variety of subterranean biotopes, especially interstitial habitats. While the family is characterized by considerable sexual dimorphism, this dimorphism has adversely affected our understanding of the systematics of the group. Most species have restricted geographic ranges and occur in difficult to sample habitats, so it is common for individual species descriptions to be based on a single sex. In this work we revisit an analysis of morphological characters in an attempt to clarify their phylogenetic utility in resolving taxonomic relationships among genera by introducing a new species, two additional characters, and phylogenetic statistical support values. Eobogidiella venkataramani sp. n., from a spring fed brook in the Shirawati River basin along the escarpment of the Western Ghats (Karnataka, India) differs from the only known congener, Eobogidiella purmamarcensis, from Argentina, in the structure of mouthparts, the shape and ornamentation on gnathopods and characters of the telson. Our phylogenetic analyses indicate that the available morphological characters are not sufficient to resolve phylogenetic relationships within Bogidiellidae, thus these characters alone cannot be used to determine the phylogenetic placement of Eobogidiella venkataramani sp. n. within the family. Nevertheless, Eobogidiella venkataramani sp. n. shares diagnostic characters with Eobogidiella, supporting placement of the new species in this genus. Our findings point towards a critical need to resolve relationships within the family using molecular approaches, along with the development of a suite of additional morphological characters for Bogidiellidae. This is the third species of Bogidiellidae from southern India.

Molecular systematics and biodiversity of oniscidean isopods in the groundwater calcretes of central Western Australia.

Groundwater calcrete aquifers of central Western Australia have been shown to contain a high diversity of stygobiont (subterranean aquatic) invertebrates, with each species confined to an individual calcrete and the entire system resembling a 'subterranean archipelago' containing hundreds of isolated calcretes. Here, we utilised alternative sampling techniques above the water table and uncovered a significant fauna of subterranean terrestrial oniscidean isopods from the calcretes. We explored the diversity and evolution of this fauna using molecular analyses based on one mitochondrial gene, Cytochrome C Oxidase Subunit I (COI), two Ribosomal RNA genes (28S and 18S), and one protein coding nuclear gene, Lysyl-tRNA Synthetase (LysRS). The results from 12 calcretes showed the existence of 36 divergent DNA lineages belonging to four oniscidean families (Paraplatyarthridae, Armadillidae, Stenoniscidae and Philosciidae). Using a combination of phylogenetic and species delimitation methods, we hypothesized the occurrence of at least 27 putative new species of subterranean oniscideans, of which 24 taxa appeared to be restricted to an individual calcrete, lending further support to the "subterranean island hypothesis". Three paraplatyarthrid species were present on adjacent calcretes and these exceptions possessed more ommatidia and body pigments compared with the calcrete-restricted taxa, and are likely to represent troglophiles. The occurrence of stenoniscid isopods in the calcretes of central Western Australia, a group previously only known from the marine littoral zone, suggests a link to the marine inundation of the Eucla basin during the Late Eocene. The current oniscidean subterranean fauna consists of groups known to be subtropical, littoral and benthic, reflecting different historical events that have shaped the evolution of the fauna in the calcretes.