Heteroonops (Araneae, Oonopidae) spiders from Hispaniola: the discovery of ten new species

Abstract The Caribbean biodiversity hotspot harbors vast reserves of undiscovered species. A large-scale inventory of Caribbean arachnids (CarBio) is uncovering new species across the arachnid tree of life, and allowing inference of the evolutionary history that has generated this diversity. Herein we describe ten new species of Heteroonops (Oonopidae, or goblin spiders), from Hispaniola: H. scapulasp. nov., H. jurassicussp. nov., H. aylinalegreaesp. nov., H. verrucasp. nov., H. renebarbaisp. nov., H. yumasp. nov., H. carlosviquezisp. nov., H. gabrielsantosisp. nov., H. solanllycarreroaesp. nov. and H. constanzasp. nov. The occurrence of the pantropical type species Heteroonops spinimanus (Simon, 1891) is reported and new localities are given for: H. validus (Bryant, 1948), H. vega (Platnick & Dupérré, 2009) and H. castelloides (Platnick & Dupérré, 2009). Molecular phylogenies indicate substantial genetic divergence separating these taxa. This work adds to evidence that the depth of diversity in the Caribbean biodiversity hotspot is particularly striking for tiny taxa living in leaf litter.


Introduction
The Greater Antilles islands form the most species-rich landmasses in the Caribbean biodiversity hotspot. These islands serve as exceptional systems for studies of species formation and biogeography (Ricklefs and Bermingham 2008). Our ongoing large-scale inventory of Caribbean arachnids (CarBio) is rapidly uncovering new species across the arachnid tree of life and offering new insight into Caribbean biogeography (e.g., Dziki et al. 2015;Agnarsson et al. 2018;Chamberland et al. 2018;Čandek et al. 2019;Tong et al. 2019;Čandek et al. 2020). Yet the biodiversity of many of these islands, including Hispaniola, remains poorly known, especially with respect to tiny cryptic arthropods, such as oonopid spiders. The family Oonopidae currently includes 1846 species distributed in 113 genera, making it the 8 th largest spider family (World Spider Catalog 2020). In 2006, the Planetary Biodiversity Inventory (PBI, 2020) project on Oonopidae was launched. At the time only 459 species of Oonopidae were known (PBI, 2020). In eleven years, the PBI project led to the discovery and descriptions of nearly 1300 new oonopid species, increasing our knowledge of the fauna by 300%. Yet, new species continue to be discovered as new areas are more thoroughly sampled, such as during the ongoing Caribbean arachnid biodiversity inventory (project CarBio).
Oonopidae are small (1.0-5.0 mm) yellow, orange to bright red haplogyne spiders. Most members of this family are found living in leaf litter, but some live in canopies (Fannes et al. 2008, Platnick andDupérré 2011b) or caves (Chamberlin and Ivie 1938), and some are termite nest inquilines (Benoit 1964) or even ant-mimics Platnick and Dupérré 2011b). Oonopids typically have six large contiguous eyes (Ubick 2005), but some species have only two (Platnick 2000), or lack eyes altogether (Chamberlin and Ivie 1938;Benoit 1964;Baehr and Ubick 2010). Oonopids show other striking morphological features, including some with elongated carapace prongs (Abrahim et al. 2012), clypeal prongs (Platnick and Dupérré 2011a) and various cheliceral and endite modifications (e.g., Kranz-Baltensperger 2012;Tong et al. 2018). But an even more peculiar morphological feature is the occurrence of male palpal asymmetry, extremely rare in spiders (Huber et al. 2007), but found in oonopid genera such as Escaphiella, Paradysderina (Platnick andDupérré 2009, 2011c). In Paradysderina the left and right male palps are so different that if observed independently, even experienced taxonomists would consider them to belong to distinct species (Platnick and Dupérré 2011c). Platnick and Dupérré (2009) revised the genus Heteroonops, including 14 species, of which 10 were new. The type species of the genus, Heteroonops spinimanus (Simon, 1892), is pantropical, while the remainder of the group has a circum-Caribbean distribution, occurring from Mexico to Dominica (Platnick and Dupérré 2009). In 2009, four species were known to occur in Dominican Republic: Heteroonops castelloides (Platnick & Dupérré, 2009), H. iviei (Platnick & Dupérré, 2009), H. validus (Bryant, 1948) and H. vega (Platnick & Dupérré, 2009). Here we describe ten new species and report for the first time the presence of the pantropical genotype, H. spinimanus, as well as new localities for H. vega, H. castelloides and H. validus. We demonstrate substantial genetic divergence between these species and analyze biogeographic patterns within Hispaniola using mitochondrial phylogenies.

Collections examined
All 66 specimens examined are from the 2012 CarBio expedition to Dominican Republic, unless otherwise noted. They were all found in leaf litter samples that were sifted in the field and either hand sorted, or extracted through Berlese funnels. Specimens are stored at the Natural History Museum in Vermont, USA (UVM); type specimens are deposited at the National Museum of Natural History, Smithsonian Institution, Washington, USA (NMNH, USNMENT). Specimens were roughly sorted in-field and stored in 95% ethanol at -20 °C upon return to the laboratory. Species determination was done through morphological assessment, followed by molecular phylogenetic analyses. Genetic divergences guided further morphological assessment and final species delineation.

Morphological assessment
Specimens were collected and examined in 95% ethanol under a SMZ-U Nikon dissection microscope. A Nikon Coolpix 950 digital camera attached to the microscope was used to photograph all the structures to be illustrated. The digital photos were used to trace proportions and the illustrations were detailed and shaded by referring back to the structure under the microscope. Female genitalia were excised using a sharp entomological needle and submerged in lactic acid to clear internal structures. The structures were photographed and illustrated as explained above. All measurements are in millimeters. For complete morphological description of the genus see Platnick and Dupérré (2009: 17-21). Nomenclatural morphology follows Platnick and Dupérré (2009).

Molecular analyses
DNA extraction was done with the QIAGEN DNeasy Tissue Kit (Qiagen, Inc., Valencia, CA). We sequenced fragments of the mitochondrial Cytochrome c oxidase subunit 1 (COI) and 16S ribosomal RNA (16S), which are typically effective phylogenetic markers at low taxonomic levels for spiders. We amplified COI with LCO1490-2776 and 16S with 16SF and 16SR using standard protocols (see e.g., Agnarsson et al. 2007). PCR products were sequenced at the University of Arizona, Beckman Genomics, or the Smithsonian Institution. Sequences were interpreted from chromatograms using Phred and Phrap (Green andEwing 2002, Green 2009) within the Chromaseq module (Maddison and Maddison 2020) in Mesquite 3.61 (Maddison and Maddison 2019), with default parameters. The sequences were then proofread by examining chromatograms by eye.
The taxon sampling in our final dataset included mitochondrial sequences for 37 of 38 Heteroonops from the Dominican Republic in our dataset (Table 1). We obtained COI data for all 37 of these specimens, and 16S for 32 of 37. Neither CO1 nor 16S amplified from the single representative of H. solanllycarreroae sp. nov. The concatenated alignment is 1114 nucleotides. For phylogenetic analyses, alignments were done in MAFFT (Katoh 2013) through the online portal EMBL-EBI, using default settings but increasing the tree rebuilding and maxiterate settings to 100. Gaps were treated as missing characters. The aligned sequences for COI, and 16S, were tested for the best fitting substitution model using the program Jmodeltest 2.1.7 (Darriba et al. 2012). The best models for each gene, among the 24 models available in MrBayes, were GTR+G for 16S and GTR+I+G for COI. We conducted Bayesian analyses using MrBayes V3.2.3 through the online portal CIPRES (Miller et al. 2010) on the concatenated mtDNA dataset. The Bayesian analyses ran 10,000,000 generations, sampling every 1000 generations. We used Tracer (Drummond and Rambaut 2007) to verify proper convergence of runs and sufficient sampling of priors.

ALE
anterior lateral eye PLE posterior lateral eye PME posterior median eye

Genitalia (male)
c bulb c conductor e embolus

Results
The ten new species of Heteroonops presented in this work are genetically distinct and distinguishable morphologically. They were all collected in leaf litter samples from forest or cave habitats in Hispaniola ranging from near sea level to 2983 m. Mitochondrial genetic divergences and patterns of relationships belie a deep and old history of Heteroonops on Hispaniola (Fig. 1).
Diagnosis. Males are diagnosed from all species by the combination of the following characters: constricted tip of palpal bulb and their bent embolus, wider apically, long conductor reaching the tip of the embolus (Figs 2, 3); females are diagnosed by the large, anterior wing-like projections of their internal genitalia and triangular anterior receptaculum (Fig. 5).
Other material examined. None. Distribution. Dominican Republic, La Vega Province (Fig. 40). Etymology. The specific epithet is a noun in apposition taken from the type locality, Jurassic Park, Dominican Republic.

Heteroonops jurassicus
Diagnosis. Males are distinguished from all species of the genera by the spatulashaped tip of the embolus (Fig. 7). Females are distinguished by their large funnelshaped anterior receptaculum (Fig. 9).
Distribution. Dominican Republic, La Vega Province (Fig. 40). Etymology. The specific epithet is a noun in apposition honoring local arachnologist and CarBio collaborator Aylin Alegre.

Heteroonops aylinalegreae
Diagnosis. Males are diagnosed from all Heteroonops by the combination of the following characters: embolus well sclerotized, not spatulated apically; short conductor not reaching the tip of the embolus (Fig. 11); females are diagnosed by their inverse triangular anterior receptaculum and large posterior receptaculum (Fig. 13).
Other Etymology. The specific epithet is a noun in apposition meaning wart in reference to the male palpal bulb bearing a wart-like projection.
Diagnosis. Males can be diagnosed from all species by the wart-like projection on the prolateral side of the bulb (Fig. 14); females can be diagnosed by their small heartshaped posterior receptaculum (Fig. 18).
Female: Unknown. Other material examined. None. Distribution. Dominican Republic, Hato Mayor Province (Fig. 40). Etymology. The specific name is noun in apposition taken from the type locality, San Rafael de Yuma, Dominican Republic.

Heteroonops yuma
Diagnosis. Females are distinguished from most species by the anterior receptaculum positioned on a narrow, short stalk; from H. vega by their larger anterior receptaculum projecting posteriorly (Fig. 22), not projecting in the later species (Platnick and Dupérré 2009, fig. 211).
Male: Unknown. Other material examined. None. Distribution. Dominican Republic, Duarte Province (Fig. 40). Etymology. The specific epithet is a noun in apposition honoring local arachnologist and CarBio collaborator Gabriel Santos.

Heteroonops gabrielsantosi
Diagnosis. Females can be diagnosed from all species by the arch wing-like projections of the internal genitalia and large oval posterior receptaculum (Fig. 27).

Heteroonops solanllycarreroae
Diagnosis. Females are diagnosed from all species by their posteriorly protruding epigastric scutum and their oval posterior receptaculum with folded bag-like extension (Fig. 29).

Discussion
Observed patterns in our data are consistent with a high probability that our sampling has only detected a small subset of the Moreover, a fourth new species H. scapula, was discovered in the same park at higher elevation (2983 m). Taxa from both of these localities are phylogenetically widespread reflecting an old most recent common ancestor and high levels of subsequent diversification (Fig. 1)). This contrasts with patterns seen in more dispersive Caribbean spiders that rarely have more than a single species of a given genus in one locality (e.g., Dziki et al. 2015, Čandek et al. 2019, Tong et al. 2019 Despite patterns consistent with high local diversity, there is evidence that some Heteroonops species are wide ranging. Two taxa that represent new records were collected far from their type localities in the Cordillera Central, H. castelloides, and H. validus. Interestingly both of these species have been collected in flight intercept traps (Platnick and Dupérré 2009) suggesting the potential for aerial dispersal. Additionally, one species described here, H. aylinalegreae, was collected in two separate low elevation localities on the northern and southern sides of Eastern Hispaniola. While it seems that some members of this genus are capable of widespread dispersal, most notably the type species, the high levels of diversity in the Dominican Republic suggest an old presence and much speciation within West Indies, consistent with biologies that are not typically dispersal prone.