Materials and methods

Specimen collection, and isolation

The specimens (i.e., dead insects and spiders) were collected by random sampling from leaf litter or from under the rocks near the roadside in the Fodingshan Nature Reserve (27°19'48"N, 108°4'48"E), Shiqian County, Tongren City, Guizhou Province, on 11th July 2025. The samples were placed in sterile bags, kept separately in an ice box, and transported to the laboratory. Specimens were preserved in the refrigerator at 4 °C until further processing.

Mycelium or conidia are picked off from the specimen and placed onto plates of potato dextrose agar (PDA) or PDA modified by the addition of 1% w/v peptone containing 0.1 g/l streptomycin and 0.05 g/l tetracycline (Chen et al. 2019a). After fungal colonies emerged from the plated samples, a piece of mycelium from the colony edge was transferred onto new PDA plates and cultured at 25 °C for 14 days under 12 h light/12 h dark conditions (Zou et al. 2010). The holotypes and ex-types cultures were deposited at the Institute of Fungus Resources, Guizhou University (formally Herbarium of Guizhou Agricultural College; code, GZAC), Guiyang City, Guizhou, China. MycoBank numbers were obtained as outlined in MycoBank (http://www.MycoBank.org) (Crous et al. 2004).

Colony characteristics were determined on PDA cultures incubated at 25 °C for 14 days, and growth rate, presence of octahedral crystals and colony colours (surface and reverse) were observed. To investigate microscopic characteristics, a little of the mycelia was picked up from the colony and mounted in lactophenol cotton blue or 20% lactic acid solution and the asexual morphological characteristics (e.g., conidiophores, phialides or conidiogenous cells, and conidia) were observed and measured using a Leica DM4 B microscope (Leica Microsystems, Wetzlar, Germany).

DNA extraction, Polymerase Chain Reaction (PCR) amplification and nucleotide sequencing

A fungal genomic DNA extraction kit (DP2033, BioTeke Corporation) was employed for DNA extraction, following the protocol described by Liang et al. (2011). The extracted DNA was stored at –20 °C for subsequent use. PCR amplification of the target genetic loci was carried out using the following primer pairs: ITS4/ITS5 (amplifying the internal transcribed spacer region, ITS) (White et al. 1990); LR0R/LR5 (targeting the 28S large subunit ribosomal gene, LSU) (Vilgalys and Hester 1990); CRPB1/RPB1Cr (targeting the RNA polymerase II largest subunit gene, rpb1) (Castlebury et al. 2004); fRPB2-5F/fRPB2-7cR (targeting the RNA polymerase II second largest subunit gene, rpb2) (Liu et al. 1999); and 983F/2218R (amplifying the translation elongation factor 1 alpha gene, tef-1α) (Castlebury et al. 2004). The thermal cycling conditions for PCR amplification were based on the methodology outlined by Chen et al. (2021a). The resulting PCR products were purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd. All the used and newly generated sequences were deposited into GenBank, and the corresponding accession numbers were obtained (Table 1).

Species | Strain | Host/Substrate | GenBank accession no. | Reference
ITS | LSU | rpb1 | rpb2 | tef-1α
Akanthomyces aculeatus | HUA 186145T | Lepidoptera | - | MF416520 | - | - | MF416465 | Kepler et al. 2017
A. aculeatus | HUA 772 | Lepidoptera | KC519371 | KC519370 | - | - | KC519366 | Sanjuan et al. 2014
Ascopolyporus polychrous | PC 546 | Hemiptera | - | DQ118737 | DQ127236 | - | DQ118745 | Chaverri et al. 2005
Ascopolyporus albus | BCC 48975T | On dead culms of bamboo | OL331502 | OL322048 | OL322056 | OL322065 | OL322035 | Thanakitpipattana et al. 2022
A. albus | BCC 48976 | On dead culms of bamboo | OL331503 | OL322049 | OL322057 | OL322066 | OL322036 | Thanakitpipattana et al. 2022
Arachnidicola sp. | KY47341 | Spider (Araneae) | PV870563 | PV870565 | - | - | PV865556 | This study
Arachnidicola sp. | KY47342 | Spider (Araneae) | PV870564 | PV870566 | - | - | PV865557 | This study
A. araneicola | GY29011T | Spider (Araneae) | MK942430 | - | MK955944 | MK955947 | MK955950 | Chen et al. 2019b
A. araneicola | GY29012 | Spider (Araneae) | MK942435 | - | MK955945 | MK955948 | MK955951 | Chen et al. 2019b
A. araneogena | GZUIF DX1 | Spider (Araneae) | KU893152 | - | MH978181 | MH978184 | - | Chen et al. 2018
A. araneogena | GZUIF SN1 | Spider (Araneae) | MH978177 | - | MH978183 | MH978186 | MH978188 | Chen et al. 2018
A. bashanensis | CQ05621T | Spider (Araneae) | OQ300412 | OQ300420 | - | OQ349684 | OQ325024 | Chen et al. 2023
A. bashanensis | CQ05622 | Spider (Araneae) | OQ300411 | OQ300421 | - | OQ349685 | OQ325025 | Chen et al. 2023
A. beibeiensis | CQ05921T | Spider (Araneae) | OQ300415 | OQ300424 | - | OQ349688 | OQ325028 | Chen et al. 2023
A. beibeiensis | CQ05922 | Spider (Araneae) | OQ300416 | OQ300427 | - | OQ349689 | OQ325029 | Chen et al. 2023
A. carrolliae | MST-FP3895T | Dead Insecta | PX368950 | PX352470 |  | PX380462 | PX380463 | Tan et al. 2025
A. hookerae | MST-FP3877T | Dead Insecta | PX368951 | PX353450 |  | PX380464 | PX380465 | Tan et al. 2025
A. fodingshanensis | SQ57131T | Spider (Araneae) | PX506041 | PX506049 | PX549495 | PX549501 | PX549505 | This study
A. fodingshanensis | SQ57132 | Spider (Araneae) | PX506042 | PX506050 | PX549496 | PX549502 | PX549506 | This study
A. kanyawimiae | TBRC 7242 | Spider (Araneae) | MF140751 | MF140718 | MF140784 | MF140808 | MF140838 | Mongkolsamrit et al. 2018
A. kanyawimiae | TBRC 7244T | Spider (Araneae) | MF140752 | MF140716 | - | - | MF140836 | Mongkolsamrit et al. 2018
A. kunmingensis | YFCC 1708939 | Spider (Araneae) | OQ509521 | OQ509508 | OQ511533 | OQ511547 | OQ506284 | Wang et al. 2024b
A. kunmingensis | YFCC 1808940T | Spider (Araneae) | OQ509522 | OQ509509 | OQ511534 | OQ511548 | OQ506285 | Wang et al. 2024b
A. sinensis | ZY06511T | Spider (Araneae) | PV082711 | PV082832 | - | PV171173 | PV171231 | Chen et al. 2025b
A. sinensis | ZY06512 | Spider (Araneae) | PV082712 | PV082833 | - | PV171174 | PV171232 | Chen et al. 2025b
A. subaraneicola | YFCC 2107937T | Spider (Araneae) | OQ509527 | OQ509514 | OQ511539 | OQ511553 | OQ506290 | Wang et al. 2024b
A. subaraneicola | YFCC 2107938 | Spider (Araneae) | OQ509528 | OQ509515 | OQ511540 | OQ511554 | OQ506291 | Wang et al. 2024b
A. sulphurea | TBRC 7248T | Spider (Araneae) | MF140758 | MF140722 | MF140787 | MF140812 | MF140843 | Mongkolsamrit et al. 2018
A. sulphurea | TBRC 7249 | Spider (Araneae) | MF140757 | MF140721 | MF140786 | MF140734 | MF140842 | Mongkolsamrit et al. 2018
A. thailandica | TBRC 7245T | Spider (Araneae) | MF140754 | - | - | MF140809 | MF140839 | Mongkolsamrit et al. 2018
A. thailandica | TBRC 7246 | Spider (Araneae) | MF140755 | MF140719 | - | MF140810 | MF140840 | Mongkolsamrit et al. 2018
A. tiankengensis | KY11571T | Spider (Araneae) | ON502848 | ON502825 | - | ON525446 | ON525447 | Chen et al. 2022a
A. tiankengensis | KY11572 | Spider (Araneae) | ON502821 | ON502827 | - | ON525448 | ON525449 | Chen et al. 2022a
A. waltergamsii | TBRC 7250 | Spider (Araneae) | MF140749 | MF140715 | - | - | MF140835 | Mongkolsamrit et al. 2018
A. waltergamsii | TBRC 7251 | Spider (Araneae) | MF140747 | MF140713 | MF140781 | MF140805 | MF140833 | Mongkolsamrit et al. 2018
A. zunyiensis | ZY06061T | Spider (Araneae) | PV082713 | PV082834 | - | PV171175 | PV171233 | Chen et al. 2025b
A. zunyiensis | ZY06062 | Spider (Araneae) | PV082714 | PV082835 | - | PV171176 | PV171234 | Chen et al. 2025b
Beauveria bassiana | ARSEF 1564T | Lepidoptera | HQ880761 | - | HQ880833 | HQ880905 | HQ880974 | Rehner et al. 2011
B. brongniartii | ARSEF 617 | Coleoptera | HQ880782 | - | HQ880854 | - | HQ880991 | Rehner et al. 2011
Kanoksria zaquensis | HMAS 246915T | Ophiocordyceps sinensis | MT789699 | MT789697 | MT797810 | - | MT797812 | Wang et al. 2023
K. zaquensis | HMAS 246917 | Ophiocordyceps sinensis | MT789698 | MT789696 | MT797809 | - | MT797811 | Wang et al. 2023
Purpureocillium lilacinum | CBS 431.87 | Soil | AY624188 | EF468844 | EF468897 | EF468940 | EF468791 | Sung et al. 2007
P. lilacinum | CBS 284.36T | Soil | AY624189 | FR775484 | EF468898 | EF468941 | EF468792 | Sung et al. 2007
Simplicillium album | FZ3638 | Soil | MK329135 | - | - | - | - | Zhang et al. 2021
S. album | CGMCC 3.19635T | Soil | NR_172844 | NG_075278 | - | - | MK336068 | Zhang et al. 2021
S. album | FZ3352 | Soil | MK329134 | - | - | - | - | Zhang et al. 2021
S. aogashimaense | JCM 18167T | Soil | AB604002 | LC496874 | - | - | LC496904 | Kondo et al. 2020
S. aogashimaense | JCM 18168 | Soil | AB604004 | LC496875 | - | - | - | Kondo et al. 2020
S. araneae | DY101811T | Spider (Araneidae) | OM743774 | OM743792 | - | - | OM818465 | Chen et al. 2022b
S. araneicola | DY11251T | Spider (Araneidae) | PV082783 | PV082900 | - | - | PV171301 | Chen et al. 2025b
S. araneicola | DY11252 | Spider (Araneidae) | PV082784 | PV082901 | - | - | PV171302 | Chen et al. 2025b
S. bursae | ZY06121T | Cocoon (Lepidoptera) | PV082785 | PV082902 | - | - | PV171303 | Chen et al. 2025b
S. bursae | ZY06122 | Cocoon (Lepidoptera) | PV082786 | PV082903 | - | - | PV171304 | Chen et al. 2025b
S. calcicola | LC5586T | Rock | KU746706 | KU746752 | - | - | KX855252 | Zhang et al. 2017
S. calcicola | LC5371 | Rock | KU746705 | KU746751 | - | - | KX855251 | Zhang et al. 2017
S. cantharise | ZY06421T | Beetle (Coleoptera) | PV082787 | PV082904 | - | - | PV171305 | Chen et al. 2025b
S. cantharise | ZY06422 | Beetle (Coleoptera) | PV082788 | PV082905 | - | - | PV171306 | Chen et al. 2025b
S. cicadellidae | GY11011T | Leafhopper (Hemiptera) | MN006243 | - | MN022271 | - | MN022263 | Chen et al. 2019a
S. cicadellidae | GY11012 | Leafhopper (Hemiptera) | MN006244 | - | MN022272 | - | MN022264 | Chen et al. 2019a
S. coccinellidae | DY101791T | Sacrab (Coccinellidae) | MT453861 | - | - | - | MT471341 | Chen et al. 2021b
S. coccinellidae | DY101792 | Sacrab (Coccinellidae) | MT453864 | - | - | - | MT471342 | Chen et al. 2021b
S. coleopterorum | SD05381T | Beetle (Coleoptera) | OM743920 | OM743925 | - | - | OM818467 | Chen et al. 2022b
S. coleopterorum | SD05382 | Beetle (Coleoptera) | OM744109 | OM744170 | - | - | OM818468 | Chen et al. 2022b
S. cylindrosporum | JCM 18169T | Soil | AB603989 | LC496876 | - | - | LC496906 | Kondo et al. 2020
S. cylindrosporum | JCM 18170 | Soil | AB603994 | LC496877 | - | - | LC496907 | Kondo et al. 2020
S. cylindrosporum | JCM 18171 | Soil | AB603997 | - | - | - | - | Kondo et al. 2020
S. cylindrosporum | JCM 18172 | Soil | AB603998 | - | - | - | - | Kondo et al. 2020
S. cylindrosporum | JCM 18173 | Soil | AB603999 | - | - | - | - | Kondo et al. 2020
S. fodingshanense | SQ57101T | Cocoon (Lepidoptera) | PX506045 | PX506053 | PX549499 | - | PX549509 | This study
S. fodingshanense | SQ57102 | Cocoon (Lepidoptera) | PX506046 | PX506054 | PX549500 | - | PX549510 | This study
S. formicae | MFLUCC 181379T | Formicidae | MK766511 | MK766512 | - | - | MK926451 | Wei et al. 2019
S. formicidae | DL10041T | Formicidae | MN006241 | - | - | - | - | Chen et al. 2019a
S. formicidae | DL10042 | Formicidae | MN006242 | - | - | - | - | Chen et al. 2019a
S. guizhouense | DY10051T | Formicidae | OM743225 | OM743226 | - | - | OM818453 | Chen et al. 2022b
S. guizhouense | DY10052 | Formicidae | OM743241 | OM743252 | - | - | OM818454 | Chen et al. 2022b
S. humicola | CGMCC 3.19573T | Soil | NR_172845 | NG_075279 | - | - | MK336071 | Zhang et al. 2021
S. humicola | LC 12494 | Soil | - | - | - | - | MK336072 | Zhang et al. 2021
S. hymenopterorum | DY101691T | Ant (Hymenoptera) | MT453848 | - | MT471344 | - | MT471337 | Chen et al. 2021b
S. hymenopterorum | DY101692 | Ant (Hymenoptera) | MT453851 | - | - | - | MT471338 | Chen et al. 2021b
S. lamellicola | CBS 116.25T | Agaricus bisporus | AJ292393 | AF339552 | DQ522404 | - | DQ522356 | Spatafora et al. 2007; Sung et al. 2001
S. lamellicola | KYK00006 | - | AB378533 | - | - | - | - | Nonaka et al. 2013
S. lamellicola | UAMH 2055 | - | AF108471 | - | - | - | - | Sung et al. 2001
S. lamellicola | UAMH 4785 | - | AF108480 | - | - | - | - | Sung et al. 2001
S. lanosoniveum | CBS 704.86 | Hemileia vastatrix | AJ292396 | AF339553 | DQ522406 | - | DQ522358 | Spatafora et al. 2007; Sung et al. 2001
S. lanosoniveum | CBS 123.42T | - | NR_171734 | NG_068571 | - | - | - | Vu et al. 2019
S. larvatum | DY101731T | Lepidoptera | OM743438 | OM743441 | OM818460 | - | OM818462 | Chen et al. 2022b
S. larvatum | DY101732 | Lepidoptera | OM743454 | OM743485 | - | - | OM818464 | Chen et al. 2022b
S. lepidopterorum | GY29131T | Lepidoptera | MN006246 | - | MN022273 | - | MN022265 | Chen et al. 2019b
S. lepidopterorum | GY29132 | Lepidoptera | MN006245 | - | MN022274 | - | MN022266 | Chen et al. 2019b
S. minatense | JCM 18176T | Soil | AB603992 | - | - | - | - | Nonaka et al. 2013
S. minatense | JCM 18177 | Soil | AB603991 | - | - | - | - | Nonaka et al. 2013
S. minatense | JCM 18178 | Soil | AB603993 | - | - | - | - | Nonaka et al. 2013
S. neoaraneae | ZY06261 T | Spider (Araneae) | PV082789 | PV082906 | - | - | PV171307 | Chen et al. 2025b
S. neoaraneae | ZY06262 | Spider (Araneae) | PV082790 | PV082907 | - | - | PV171308 | Chen et al. 2025b
S. neocoleopterorum | DY091481T | Ladybug (Coleoptera) | OR121066 | OR121065 | - | - | OR126575 | Chen et al. 2024
S. neocoleopterorum | DY091482 | Ladybug (Coleoptera) | OR121064 | OR121067 | - | - | OR126576 | Chen et al. 2024
S. neolepidopterorum | DY101751T | Lepidoptera | MT453854 | - | - | - | MT471339 | Chen et al. 2021b
S. neolepidopterorum | DY101752 | Lepidoptera | MT453857 | - | - | - | MT471340 | Chen et al. 2021b
S. niveum | BCC83036T | Ophiocordyceps camponoti-leonardi | MW621499 | MW620992 | - | - | MW603488 | Crous et al. 2021
S. obclavatum | CBS 311.74T | Air | AJ292394 | AF339517 | - | - | EF468798 | Sung et al. 2007
S. obclavatum | JCM 18179 | Soil | AB604000 | - | - | - | - | Nonaka et al. 2013
S. pechmerlense | CBS 147188T | Air | MW031272 | MW031268 | MW033222 | - | MW033224 | Leplat et al. 2021
S. puwenense | YFCC 23129490T | Spider (Araneae) | PQ508796 | PQ508802 | PQ560994 | - | PQ537122 | Lu et al. 2025
S. puwenense | YFCC 23069492 | Spider (Araneae) | PQ508798 | PQ508803 | PQ560995 | - | PQ537124 | Lu et al. 2025
S. salviniae | NTUPPMCC 20-074T | Salvinia auriculata | MT974200 | MT974415 | - | - | MW200240 | Chuang et al. 2024
S. salviniae | NTUPPMCC 20-075 | Salvinia auriculata | MT974201 | MT974416 | - | - | MW200241 | Chuang et al. 2024
S. scarabaeoidea | DY101391T | Scarabaeoidea | MT453842 | - | MT471343 | - | MT471335 | Chen et al. 2021b
S. scarabaeoidea | DY101392 | Scarabaeoidea | MT453845 | - | - | - | MT471336 | Chen et al. 2021b
S. shiqianense | SQ57111T | Spider (Araneae) | PX506047 | PX506055 | - | - | PX549511 | This study
S. shiqianense | SQ57112 | Spider (Araneae) | PX506048 | PX506056 | - | - | PX549512 | This study
S. sinense | AFMCCC 16aT | Human skin | OQ332403 | - | - | - | OQ352167 | Yan et al. 2023
S. sinense | AFMCCC 16b | Human skin | OQ332404 | - | - | - | OQ352168 | Yan et al. 2023
S. spumae | JCM 39051T | Soil | LC496870 | LC496884 | - | - | LC496914 | Kondo et al. 2020
S. spumae | JCM 39050 | Soil | LC496869 | LC496883 | - | - | LC496913 | Kondo et al. 2020
S. spumae | JCM 39054 | Soil | LC496871 | LC496887 | - | - | LC496917 | Kondo et al. 2020
S. subtropicum | JCM 18180T | Soil | AB603990 | LC496880 | - | - | LC496910 | Kondo et al. 2020
S. subtropicum | JCM 18181 | Soil | AB603995 | LC496881 | - | - | LC496911 | Kondo et al. 2020
S. subtropicum | JCM 18182 | Soil | AB603996 | - | - | - | - | Kondo et al. 2020
S. subtropicum | JCM 18183 | Soil | AB604001 | - | - | - | - | Kondo et al. 2020
S. sympodiophorum | JCM 18184T | Soil | AB604003 | LC496882 | - | - | LC496912 | Kondo et al. 2020
S. vallense | DY09921T | Spider (Araneae) | OR121058 | OR121059 | - | - | OR126573 | Chen et al. 2024
S. vallense | DY09922 | Spider (Araneae) | OR121060 | OR121061 | - | - | OR126574 | Chen et al. 2024
S. wudangense | WD04131 T | Spider (Araneae) | PV082791 | PV082908 | PV171163 | - | PV171309 | Chen et al. 2025b
S. wudangense | WD04132 | Spider (Araneae) | PV082792 | PV082909 | PV171164 | - | PV171310 | Chen et al. 2025b
S. yunnanense | YFCC 7133T | Akanthomyces waltergamsii | - | MN576784 | MN576844 | - | MN576954 | Wang et al. 2020
S. yunnanense | YFCC 7134 | Akanthomyces waltergamsii | - | MN576785 | MN576845 | - | MN576955 | Wang et al. 2020
S. zunyiense | ZY06581T | Spider (Araneae) | PV082793 | PV082910 | - | - | PV171311 | Chen et al. 2025b
S. zunyiense | ZY06582 | Spider (Araneae) | PV082794 | PV082911 | - | - | PV171312 | Chen et al. 2025b

Sequence alignments and phylogenetic analyses

DNASTAR Lasergene (v 6.0) was used to edit DNA sequences in this study. We carried out two phylogenetic analyses to confirm the strains in different taxonomic hierarchies. The reference sequences of ITS, LSU, rpb1, rpb2 and tef-1α sequences for these analyses were downloaded from GenBank according to Chen et al. (2025b) and Chang et al. (2026), with additional sequences chosen through BLASTn searches. All the sequences were aligned and edited by MAFFT v.7.487 (Katoh and Standley 2013) and MEGA6 (Tamura et al. 2013).

To determine the phylogenetic placement of the novel isolates, two independent analyses were performed using concatenated gene datasets – the first included ITS, LSU, rpb1, rpb2 and tef-1α to infer relationships within Arachnidicola s. str. (Dataset 1), while the second employed ITS, LSU, rpb1 and tef-1α to ascertain the position within Simplicillium s. str. (Dataset 2).

Both concatenated datasets were assembled using SequenceMatrix v.1.7.8 (Vaidya et al. 2011), and the optimal substitution models for Bayesian analysis were determined using ModelFinder (Kalyaanamoorthy et al. 2017) implemented in PhyloSuite v.1.2.2 (Zhang et al. 2020). The two datasets were then subjected to identical phylogenetic analyses using both Bayesian inference (BI) and maximum likelihood (ML) methods. For BI, MrBayes v.3.2 (Ronquist et al. 2012) was run for 10,000,000 generations under a Markov chain Monte Carlo (MCMC) algorithm, sampling every 500th tree to yield 20,001 trees; the first 4,000 trees were discarded as burn-in, and the remaining 16,001 trees were used to compute posterior probabilities on the 50% majority-rule consensus tree. After the analysis was finished, Tracer v.1.5 (Drummond and Rambaut 2007) was used to verify that the effective sample size (ESS) for all parameters was greater than 200, confirming the convergence of both runs and the adequacy of the burn-in.

Maximum likelihood (ML) analyses were performed using IQ-TREE v.2.0 (Trifinopoulos et al. 2016). The optimal substitution models for each partition were automatically selected by the built-in ModelFinder function (Kalyaanamoorthy et al. 2017) based on the Bayesian information criterion (BIC). Nodal support was assessed using ultrafast bootstrap approximation (UFBoot) with 1,000 replicates (Minh et al. 2013), and the Shimodaira–Hasegawa-like approximate likelihood ratio test (SH-aLRT) with 1,000 replicates was also computed for branch support. The best-scoring ML tree was searched using the standard hill-climbing algorithm with 100 independent starting parsimony trees.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) was evaluated using the pairwise homoplasy index (PHI) test (Bruen et al. 2006) implemented in SplitsTree4 v.4.16.1 (Huson and Bryant 2006). The analysis was performed separately for each genus using concatenated datasets of three gene loci (ITS, LSU and tef-1α), with 1,000 bootstrap replicates, following the protocol described by Quaedvlieg et al. (2014). For Arachnidicola, the dataset included 11 taxa (comprising the new species A. fodingshanensis and its closely related species), while for Simplicillium, the dataset comprised four taxa (including S. fodingshanensis, S. shiqianense, and their close relatives). The PHI test detects recombination by evaluating the level of phylogenetic conflict among loci; a Φw value below 0.05 indicates significant recombination (suggesting that the tested taxa are not genetically distinct), whereas a value above 0.05 indicates no significant recombination, supporting the recognition of distinct species (Chaiwan et al. 2022). Additionally, single-gene phylogenies were compared with the concatenated-gene analysis to assess congruence among loci; the absence of conflicting topologies among individual gene trees and the concatenated tree provides further support for species delimitation. A split graph was constructed using the LogDet transformation (Lockhart et al. 1994) together with the split decomposition method (Bandelt and Dress 1992) to visualize the phylogenetic signals and potential recombination events among the sampled taxa.