Research Article |
Corresponding author: Zhengyang Wang ( zhengyangwang@g.harvard.edu ) Academic editor: Erik J. van Nieukerken
© 2019 Zhengyang Wang, Hailing Zhuang, Min Wang, Naomi E. Pierce.
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.
Citation:
Wang Z, Zhuang H, Wang M, Pierce NE (2019) Thitarodes shambalaensis sp. nov. (Lepidoptera, Hepialidae): a new host of the caterpillar fungus Ophiocordyceps sinensis supported by genome-wide SNP data. ZooKeys 885: 89-113. https://doi.org/10.3897/zookeys.885.34638
|
A new species of ghost moth, Thitarodes shambalaensis sp. nov., is described from Yanzigou glacier, Mt. Gongga, Sichuan, China. The species is a host of the economically important caterpillar fungus Ophiocordyceps sinensis. Establishment of this new species is supported by morphology and genetic differentiation measured in a CO1 phylogeny and in genome-wide SNP coverage. A summary tree from 538 sequences of different genetic markers from Thitarodes (including sequences extracted from caterpillar fungus sclerotium samples) support the genus Thitarodes as a monophyletic group, and indicate that Thitarodes is the host genus for O. sinensis. Sampling efforts so far have centered on half of the known phylogenetic diversity of Thitarodes, with some species-level clusters (separated by < 2.5% genetic distance) containing 17 described species. Fifteen clusters are known from either a single “orphan taxon” or a single sequence from a caterpillar fungus sclerotium sample. We provide suggestions for building a more robust phylogeny of the genus Thitarodes and highlight some of the conservation threats that species from this genus face due to unprecedented habitat exploitation.
RAD-Seq, phylogeny, new species, caterpillar fungus, Kangding, Mt. Gongga, Sichuan, China
The genus Thitarodes Viette, 1968 of the ghost moth family Hepialidae was first established by Viette,1968 to accommodate Hepialus armoricanus Oberthür, 1909 found in China and three other new species found in Nepal (T. danieli Viette, 1968, T. eberti Viette, 1968, and T. dierli Viette, 1968).
After
The inventory by
1. Thitarodes adults are difficult to collect. Both
2. Since most Thitarodes species have few distinctive wing scale patterns, new species descriptions are based primarily on male genitalia, sometimes coupled with a description of wing venation. Moths of the infraorder Exoporia, which contain the genus Thitarodes, are distinguished by the unique configuration of female genitalia: in female exoporian Lepidoptera, sperm is transferred to the egg via an external seminal gutter. This important feature has been largely ignored in descriptions of the genus Thitarodes. After re-examining the holotype of T. armoricanus,
3. Several Thitarodes species described before 2000s were only known by drawings of wing venation and genitalia structure. Access to the holotypes of these species is limited. Many species are only known from a few samples collected in restricted localities (such as T. zhongzhiensis (Liang, 1995) in “the middle of Renzhi snow mountain”, T. anomopterus (Yang, 1994) on the “north-west slope of Mt. Laojun, Yunnan” , T. jialangensis (Yang, 1994) in “Jialang county west of Meili mountain”), and have not been further studied (
In recent decades, molecular evidence has helped resolve difficult problems in taxonomy, including elucidating the backbone of non-ditrysian Lepidoptera including Hepialidae (
While molecular work using samples of caterpillar fungus sclerotia has only yielded at most three DNA fragments with which to evaluate each sample, the development of next-generation sequencing techniques has offered researchers new genotyping-by-sequencing methods to obtain large numbers of loci from non-model organisms for phylogenetic studies (
In this paper we describe T. shambalaensis, a new species in the genus Thitarodes from the Yanzigou glacial valley, Mt. Gongga, Sichuan, China, including analysis of wing venation, male genitalia, labial palps, phenology and habitat. We discuss some of the conservation threats this species is facing due to recent habitat exploitation related to local experimentation on caterpillar fungus-farming. We evaluate the validity of T. shambalaensis as a new species by providing data from morphology, CO1 sequences (i.e., constructing a phylogenetic tree with all known CO1 sequences of Thitarodes) and genome-wide SNP sequences (i.e., comparing intra and inter-species SNP sequence coverage), and we infer a “summary tree” from all known sequences of different genetic segments of Thitarodes, both from sequence deposits with known species names and from sequences of caterpillar fungus sclerotia (Suppl. material
All adult samples described were collected between June and July 2016, at Yangliuping (29°41'2.54"N, 101°53'32.24"E), inside Yanzigou glacier, Mt. Gongga, Sichuan (Fig.
Genomic DNA of 134 samples of Thitarodes were extracted from leg (adult) or thoracic (larvae, pupae) tissue with Qiagen DNeasy Blood and Tissue Kits (Qiagen Inc.). The COI region of 48 samples was amplified and sequenced with LCO1490 (
A total of 541 available sequences of Thitarodes was used to build a summary tree. These sequences came from three sources: CO1 and mitogenome sequences of known Thitarodes species used in generating the Maximum Likelihood CO1 tree in this study (Suppl. material
Holotype : CHINA • ♂; Mt. Gongga, Luding county, at the head of Yanzigou valley (燕子沟), Yangliuping habitat (杨柳坪); 29°41'2.54"N, 101°53'32.24"E; alt. 3892 m; 25–30 Jun. 2016; H. Zuo leg.; MK226958; Sichuan Plant Quarantine Station.
Paratypes : CHINA • 2 ♂; same collection data as for holotype • 2 ♂; Mt. Gongga, Luding county, at the head of Yanzigou valley (燕子沟), Haizidang habitat (海子凼); 29°40'17.18"N, 101°53'48.25"E; alt. 3977 m; 25–30 Jun. 2016; H. Zuo leg. • 2 ♀; same collection data as for holotype.
From the Sanskrit word शम्भल (Shambala). In Hindu and Tibetan Buddhist tradition, the term refers to a mythical kingdom hidden in the snow mountains. The name refers to the magnificence of the species’ alpine habitat under Mt. Gongga.
(based on Holotype). General. In resting position, forewings fold perpendicular above the sagittal plane of the body and abdomen, completely covering the hindwings. The apex and termen of the two forewings contact each other, while the costa of each forewing extends from the tegula, forming an isosceles. Setae above the thorax form a triangular patch. Wingspan: 44.0 mm (mean = 41.2 mm, SD = 3.5, N = 6). Forewing length: 19.6 mm (mean = 19.2 mm, SD = 1.6, N = 6), width: 9.9 mm (mean = 9.9 mm, SD = 0.6, N = 6). Hindwing length: 16.9 mm (mean = 16.5 mm, SD = 0.8, N = 6), width: 12.25 mm (mean = 11.7 mm, SD = 0.9, N = 6). Body length: 15.3 mm (mean = 14.7 mm, SD = 1.1, N = 6). Thorax width: 3.7 mm (mean = 3.6 mm, SD = 0.2, N = 6).
A Natural habitat of Thitarodes shambalaensis sp. nov. at Yanzigou glacier entrance point, Mt. Gongga, Sichuan, China (photograph credit Meng Li, May 2018) B disturbed habitat of Thitarodes shambalaensis sp. nov. due to excavation of T. shambalaensis pupae, at Haizidang, Yanzigou glacier (photograph credit Wenbin Ju, May 2019).
Head. Antenna (Fig.
Body. Red-brown. Dense ocherous and yellow setae on thorax. Red and black setae on lateral, ventral and caudal side of the abdomen.
Legs. Fig.
Wings. Fig.
Male genitalia. Fig.
Female genitalia
(based on paratype). Fig.
This species has no distinct external sexual dimorphism. Male pseudotegumen triangular with heavily sclerotized pseudotegumenal arms. Pseudotegumenal arms fan-shaped. Valva densely haired with sclerotized, hook-like apex. Venation of T. shambalaensis is similar to that of T. namnai Maczey, 2010 in Nepal, but in T. shambalaensis both A and CuP reach dorsum margin on the hindwing. Thitarodes markamensis (Liang, Li & Shen, 1992) has also reached the degree of heavy sclerotization on the pseudotegumenal arms, but the two species can be distinguished by differences in venation (where MR furcation in T. shambalaensis is distal to the furcation Rs3 and Rs4) and an inflation on the posterior margin of the saccus. Sclerotization at the ventral base of the valvae with a spinal projection is also present in other species of Thitarodes, such as in T. jiachaensis Zou, 2011 and T. sejilaensis Zou, 2011, but the spinal projection is less curved and the setose lobe of the dorsal side of the valvae is more elongated in T. shambalaensis.
With the exception of those by
Late instar larvae, sometimes already infected by O. sinensis, can be found as early as mid-May, about 30 cm under soil. Pupae can be found starting early June in soil. Adults appear in a week in late June.
The species is found in several high elevation (3400–3800 m) alpine grassland along the glaciers of eastern Mt. Gongga (Fig.
The species is the host of O. sinensis. Caterpillar fungus collection has for decades provided income for local people in eastern Mt. Gongga. The traditional method of collection has not had a discernible impact on populations of T. shambalaensis, but since 2016, medical pharmaceutical companies have begun buying T. shambalaensis pupae from local people for a commercial caterpillar fungus farming project. Many T. shambalaensis pupae have been excavated from their habitat each year, sold and transferred to commercial breeding stations. Local people have expressed concern at such habitat exploitation. When we visited the Haizidang habitat in 2019, this alpine grassland had been completely uprooted due to pupae excavation (Fig.
COI sequences of 48 Thitarodes samples collected from six glacial valleys were successfully sequenced (Figs
A total of 128 out of 134 samples from seven glacial valleys was successfully sequenced according to the RAD-seq protocol (
A Maximum Likelihood tree of CO1 sequences of species sampled in this study (yellow, red and blue dots) and other known species of the genus Thitarodes (grey dots). Outgroups are in black dots. The corresponding position of the genus Ahamus designated by
A total of 538 sequences (excluding three sequences from non-Thitarodes outgroups) was used to construct a summary phylogenetic tree (Suppl. material
Summary tree for all known species of Thitarodes. Samples in this study are labeled in color bars (blue, red and yellow). Each tip represents a cluster of all samples that are within 2.5% genetic distance from each other, the length of the grey bar represents the number of individual samples falling into the cluster. Numbers after the dots enumerate the number of samples that fall into a particular sequence type. All named individual samples are labeled. The genus Ahamus as designated by
Our analysis shows that T. shambalaensis is not only morphologically, but also phylogenetically distinct from other known species in the genus Thitarodes. Even according to the generic description by
Some other Thitarodes species also possess triangular pseudotegumina with fan-shaped, indented sclerotization at the pseudotegumenal arms, such as T. jialangensis (Yang, 1994), T. pratensis (Yang, 1994), T. callinivalis (Liang, 1995), T. litangensis (Liang, 1995), T. kangdingensis (Chu & Wang, 1985), and T. markamensis (Liang, Li & Shen, 1992). Although morphologically distinct from T. shambalaensis (e.g., T. jialangensis Yang, 1994 has a dark forewing without spots; T. pratensis has an elongated 23rd antenna segment and orange eyes), it is possible that these species, along with T. shambalaensis, are subspecific variations of a single widespread species ranging across the Hengduan Mountains. This hypothesis could not be further tested without a thorough revision of the genus.
Our phylogenies are largely consistent with the subdivision of the genus into Thitarodes and Ahamus by
Phylogenetic placement and SNP coverage visualization from our study both suggest that some larvae and pupae samples collected from a lower elevation habitat in Gangbogeng valley across two consecutive years belong to an undescribed taxon (T. shambalaensis has been collected along this same valley, but in a habitat at a higher altitude). Our attempts to collect adults of this unknown taxon from 2016 to 2018 have not been successful, nor is it found in any other valley that we sampled. The limited range of this taxon compared to the parapatric and relatively widespread T. shambalaensis and its evolutionary history is intriguing. If the group is indeed a member of the genus Ahamus, as defined by
CO1 sequences of our samples collected at Yajiageng valley are closely related (within 2% divergence) to known sequences labeled as T. gonggaensis in the study of
According to Wang and Yuo (2011), four other species have “Kangding” as the type locality or have been recorded in “Kangding”: T. kangdingroides (Chu & Wang, 1985), T. kangdingensis (Chu & Wang, 1985), T. oblifurcus (Chu & Wang, 1985), and T. sichuanus (Chu & Wang, 1985). It is unlikely that these samples were collected at Kangding city (30°01'21.1"N, 101°57'27.6"E). Although the city is one of the most prominent trading centers for caterpillar fungus, it is located at a relatively low elevation (2,500 m) and is not a known habitat of caterpillar fungus. These species, like samples of T. gonggaensis in this study and in
The type species of the genus, T. armoricanus, is also recorded as collected (and reared) by
A common concern in any study of the population genetics of non-model organisms is whether the analyzed samples come from different populations of the same species, or whether multiple species are involved. In our study, we still need to verify whether all Thitarodes samples collected across the eastern slope of Mt. Gongga are the same species. Without molecular evidence, this is difficult to do, since adult Thitarodes samples are difficult to identify, and most samples are obtained in larval or pupal forms, which are not sufficiently taxonomically informative for species delineation.
CO1 sequences show that our samples cluster into three clades, with inter-clade divergence falling below the 3.78% commonly observed in populations within a species, and intra-group divergence falling within the 11.06 % range of species within the same genus boundary (see review of CO1 sequence divergence by
RAD‐Seq has traditionally been considered useful in population level studies, while species-level divergence would reduce the amount of shared SNP coverage across species (
We conclude that in this study, both the phylogeny based on CO1 sequences and the visualization of genome-wide SNP coverage provide evidence for the presence of three species in the eastern slopes of Mt. Gongga: T. shambalaensis occupies most valleys in the eastern slopes of Mt. Gongga; T. gonggaensis is found in Yajiageng valley; another undescribed species is isolated at a low elevation habitat at Gangbogeng valley.
Our summary tree shows that the genus Thitarodes is monophyletic. All known moth sequences extracted from caterpillar fungus sclerotia, despite the difficulty of assigning them to discrete species, nevertheless cluster within the genus Thitarodes.
Among the 538 sequences from 184 individuals of the genus Thitarodes, only 42 could be associated with a species name, and only eight of these are verified in publications. Of the 54 species summarized by
In cases where multiple sources of sequences are all labeled as the same species, species identification has shown to be quite consistent: the Cytb sequence of T. yunnanensis matches to the mitogenome of T. yunnanensis, the Cytb sequence of T. jianchuanensis clusters together with CO1 sequence of T. jianchuanensis (as both are aligned with other mitogenomes), the CO1 sequence of T. gonggaensis matches its mitogenome, and the CO1 sequence of T. renzhiensis matches its mitogenome. Slight variations exist between the segment sequences and their corresponding flanking regions on the mitogenome, but we could find no phylogenetic inconsistencies. The only exception is that the two entries of the Cytb sequence of the type species of the genus, T. armoricanus, are distinctly different. While we have reason to believe that the type specimen of this sample was collected around Kangding (
Our summary tree also reveals inadequate sampling in many clades. When clustered by a 2.5% genetic distance (what we consider to be a reasonable threshold for inter-species variation, see
The bulk of the sampling effort so far has focused on only half of the known diversity of Thitarodes. This uneven phylogenetic sampling is not simply the result of relying on sequences of caterpillar fungus sclerotia, which have no morphological species description. The uneven sampling problem extends to the phylogeny of named species as well: the reference sequences of 39 out of the 42 named species fall into one of those 30 2.5%-distance clusters. One such cluster (including T. renzhiensis, T. oblifurcus, etc.) includes 17 known species! Most of these 17 species are known by one Cytb sequence only, which might be the result of sampling a particularly invariant region of the mitogenome. However, this issue has not been brought to attention in previous attempts to summarize genetic data for the genus (
To summarize, our current understanding of the genus Thitarodes derives from three major sources:
1 The analysis of samples of caterpillar fungus sclerotia, and sometimes just Thitarodes larvae or pupae, has provided crucial information about the habitat and ecology of the species in the genus, but often fails to provide information about the morphology of the Thitarodes adult. We encourage workers in this field to include more detailed descriptions of the habitats where samples of caterpillar fungus are collected (including vegetation, climate and soil type), and make an effort to understand adult Thitarodes biology in these known habitats.
2 Many described species have types deposited in institutions around China; earlier descriptions of these samples often do not include photos of wing venation and genitalia. Revisions of these taxa with updated photos and molecular data for comparative analysis are critical.
3 New descriptions of species in the group (see
Zhengyang Wang was supported by a graduate fellowship from Harvard University Department of Organismic and Evolutionary Biology. The publication of this research is supported by a grant from the Wetmore Colles Fund, Harvard Museum of Comparative Zoology. We thank Huailiang Tang, Zulian Zhou, Ming Liu, Huaping Zuo from Yanzigou valley for field assistance; we thank Dr. Kadeem Gilbert from Harvard Museum of Comparative Zoology for providing advice on an early version of the manuscript; we thank Dr. Guren Zhang from Sun Yat-Sen University for providing CO1 sequences for T. jiachaensis, T. namensis and T. sejilaensis and offering critical advice in looking for Thitarodes moths in the field. We thank Dr. Erik J. van Nieukerken, Dr. John R. Grehan and an anonymous reviewer for reviewing this manuscript and providing invaluable comments and advice; we thank Dr. Nathalie Yonow for carefully editing this manuscript. We thank Dazhong Yan, Dr. Dong Shen and Hua Zhang from the documentary production team of “Inside Shambala” for providing footage of Thitarodes shambalaensis adult.
Table S1. Sequences used in Fig.
Data type: molecular data
Table S2. Samples sequenced shown in Fig.
Data type: molecular data
Table S3. Sequences used in Fig.
Data type: molecular data