Research Article |
Corresponding author: Royce T. Cumming ( roycecumming@gmail.com ) Academic editor: Marco Gottardo
© 2020 Royce T. Cumming, Sarah Bank, Stephane Le Tirant, Sven Bradler.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Cumming RT, Bank S, Le Tirant S, Bradler S (2020) Notes on the leaf insects of the genus Phyllium of Sumatra and Java, Indonesia, including the description of two new species with purple coxae (Phasmatodea, Phylliidae). ZooKeys 913: 89-126. https://doi.org/10.3897/zookeys.913.49044
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Within the last two years, the leaf insects of the genus Phyllium of both the islands of Java and Sumatra have been reviewed extensively based on morphological observations. However, cryptic species which cannot be differentiated morphologically may be present among the various populations. Since it has frequently been demonstrated that analyses based on molecular data can bring clarity in such cases, we conducted a phylogenetic analysis based on three genes (nuclear gene 28S and mitochondrial genes COI and 16S) from the Phyllium species of these islands. The results show distinct molecular divergence for several populations and suggest the presence of two new cryptic species, morphologically inseparable from Phyllium hausleithneri Brock, 1999. From Sumatra, the population originally thought to be a range expansion for Phyllium hausleithneri, is now here described as Phyllium nisus sp. nov., with the only consistent morphological difference being the color of the eggs between the two populations (dark brown in P. hausleithneri and tan in P. nisus sp. nov.). Further, an additional population with purple coxae from Java was morphologically examined and found to have no consistent features to separate it morphologically from the other purple coxae species. This cryptic species from Java was however shown to be molecularly distinct from the other purple coxae populations from Sumatra and Peninsular Malaysia and is here described as Phyllium gardabagusi sp. nov. In addition, Phyllium giganteum is here officially reported from Java for the first time based on both historic and modern records of male specimens.
Cryptic species, molecular phylogeny, phasmid, taxonomy, walking leaf
Stick and leaf insects (Phasmatodea) are known for their extreme forms of crypsis by camouflaging themselves as parts of plants, with the majority of forms imitating twigs and exhibiting extremely slender and elongated bodies (
Previous studies have dealt with the Phylliidae of Indonesia in several regional works in the last few years [Wallacea:
Morphological examinations were done with a Leica ZOOM 2000 stereomicroscope. Measurements of the holotype were conducted to the nearest 0.1 mm using digital calipers. Egg orientation terminology follows
The following institutional abbreviations are used herein:
IMQC Insectarium de Montréal, Montréal, Québec, Canada
Coll RC Private collection of Royce T. Cumming, U.S.A.
Coll SLT Private collection of Stéphane Le Tirant, Canada
The following wing venation abbreviations are used in Figure
C costa
Sc subcosta
R radius
R1 first radius
Rs radial sector
M media
MA media anterior
MP media posterior
Cu cubitus
CuA cubitus anterior
CuP cubitus posterior
Cu+1AA cubitus fused with first anterior anal)
1AA–7AA anterior anal veins one through seven
1PA–5PA posterior anal veins one through five
1A first anal
We selected 18 Phyllium specimens that represent all eight species known from Java and Sumatra (see species checklist at the end for more details and Suppl. material
One dried hind leg per specimen was soaked in water before removing femoral muscle tissue. Genomic DNA was extracted from the muscle tissue with the Quick-DNATM Miniprep Plus kit (Zymo Research, Irvine, USA) and eluted in 60 µl elution buffer following the manufacturer's protocol. Using PCR, we amplified the two mitochondrial genes COI and 16S and parts of the nuclear gene 28S using primers described elsewhere (see Suppl. material
Sequences were aligned for each individual gene with ClustalW v. 2.1 (
For the 21 specimens sampled (18 Phyllium and three outgroups), we obtained 16 COI, 18 28S, and 21 16S sequences, resulting in a final concatenated dataset comprising 1758 bp (Suppl. material
Maximum likelihood tree of 18 Phyllium specimens from Java and Sumatra and three outgroup species. The phylogenetic tree was generated with IQ-TREE and rooted with Orthomeria kangi (Aschiphasmatinae). Ultrafast Bootstrap support values are given below nodes. Lineages with short branch lengths were collapsed. A and B depict the two main Phyllium clades. Purple circles indicate those species with purple colored coxae.
Phyllium pulchrifolium Audinet-Serville, 1838.
Malaysia: (Peninsular) Perak, Pahang, and Selangor States; (Bornean) Sarawak (Bintulu Division) and Sabah (Pensiangan) States. Indonesia: Sumatra, Bengkulu Province; (Borneo) West Kalimantan Province (Mount Bawang).
Indonesia: West Java Province (Mount Halimun and Mount Gede) (Figure
Phyllium giganteum is a widely distributed species ranging from Peninsular Malaysia (the type locality), to Borneo, Sumatra, and most recently Java, Indonesia, the furthest south this species is currently known to occur (
As a morphologically variable species with regards to abdominal shape and coloration (
Phyllium giganteum. A, B Dorsal view of male Phyllium giganteum from Java, Indonesia C–E live Phyllium giganteum A male from Mount Halimun, West Java, Coll SLT B antique male from the
Gryllis (Mantis) siccifolius Linnaeus, 1758.
Indonesia: Eastern Java (Nongkodjadjar [type locality]); Eastern Java, Mt. Argopuro; Western Java, Mt. Halimun (Figure
With the description of the very morphologically similar Phyllium gardabagusi sp. nov., also from Java, the numerous localities noted within
This is the only species in the clade that can easily be differentiated morphologically from other clade members. The coxae color in P. jacobsoni is white (see Figure
On average P. jacobsoni tend to be smaller individuals, but as with the other species in the clade there were significant outliers that made the range of sizes overlap with the other species significantly (Table
Morphological features compared between the members of clade B. Key: a As noted in
Feature | Phyllium jacobsoni | Phyllium hausleithneri | Phyllium nisus sp. nov. | Phyllium gardabagusi sp. nov. |
---|---|---|---|---|
Distribution | Java | Peninsular Malaysia | Sumatra | Java |
Female length [mm] | 63.5–75.0a/b | 74.6–82.8 | 70.3–79.3 | 68.4–77.3 |
Male length [mm] | 42.5–56.5a | 55.8–57.8 | 51.4–56.1 | 50.0–50.5 |
Number of teeth on the pars stridens of antennomere III in females | 40a | 44–48c | 37–44d | 34–39e |
Egg color | Tan to medium brown | Dark, rich brown | Pale tan to medium brown | Pale brown to medium brown |
Coxae color | White | Purple | Purple | Purple |
Malaysia: (Peninsular) Perak and Pahang states (Figure
Unconfirmed distributions:
Malaysia: Selangor State, Bukit Kutu. This record was noted as Phyllium siccifolium in
Singapore:
This species has been in the phasmid breeding community for many years (Figure
Holotype: ♀, Indonesia: Sumatra, Bengkulu Prov., Bengkulu District, Besuki Village: IV.2017, Local Collector. Deposited in the Montreal Insectarium type collection (Coll RC 18-157) (Figure
This population has been available within the phasmid breeding community for a number of years under the name Phyllium sp. “Bukit Daun” and has been noted as a reasonably easy species to breed in captivity (Figures
This population has already undergone extensive morphological scrutiny in
Eggs from the four species in clade B. A Phyllium hausleithneri, lateral view (Coll RC 18-002) B micropylar plate view (Coll RC 18-003), note that this individual exhibits a slightly longer micropylar plate than average which makes it appear longer than the other clade members, this is simply the upper limit of the morphological variation C Phyllium nisus sp. nov., lateral view (Coll RC 17-380) D micropylar plate view (Coll RC 17-377) E Phyllium gardabagusi sp. nov., lateral view F micropylar plate view G Phyllium jacobsoni, lateral view H micropylar plate view. Photos E–H courtesy of Bruno Kneubühler.
Phyllium nisus sp. nov. is possibly the species referred to in
Female. Coloration. Individuals are always a vibrant pale green with varying degrees of reddish or grayish brown coloration on specific regions of the body (Figures
Morphology.
Head. Head capsule about as long as wide, vertex with granulation throughout the surface, some more closely spaced than others (Figure
Representative female and male tegmina and alae wing venation present in our molecularly identified Clade B (see Figure
Male. Coloration. Overall coloration pale green throughout with variable patches of brown to reddish coloration (Figure
Morphology.
Head. Head capsule longer than wide, with a vertex that is nearly completely smooth or in some individuals there can be two or three small nodes near the posteromedial tubercle (Figure
The lateral surfaces are flattened and the dorsal surface is slightly convex, which gives the egg a slight bend (Figure
Measurements including the extended pinnae [mm]. Length (including operculum) 6.2–6.5 mm, maximum width of capsule when viewed from lateral aspect 3.6–3.8 mm, length of micropylar plate 2.5–2.6 mm.
General color throughout the body (including head and antennae) is dark brown to black (Figure
Noun, Greek in origin, Νῖσος. Named after Nisus, king of Megara, who had a single purple lock of hair that, for as long as it was not cut, guaranteed him life and possession of his kingdom. We felt that this homage was fitting to the purple-haired king as this species has the singular purple feature (coxae) which is unique among the Phylliidae with only the species in this clade known to have purple coxae.
Holotype: ♀: Indonesia: West-Java, Mt. Halimun: August 2014. Deposited in the Montreal Insectarium type collection (Coll RC 16-202) (Figure
This population has also recently entered the phasmid breeding community under the culture name of Phyllium sp. “Argopuro, Blue-coxae” (Figures
Female. Coloration. Specimens are always a vibrant pale green with varying degrees of reddish or grayish brown coloration more common on specific regions of the body. On the lightest colored individuals, no brown markings are present, with even the antennae a pale color similar to the shade of green seen on the head capsule (Figure
Morphology.
Head. Head capsule about as long as wide, vertex with granulation throughout the surface, some more closely spaced than others (Figure
Male. Coloration. Overall coloration pale green throughout with some veins and nodes a lighter yellow color (Figure
Morphology.
Head. Head capsule longer than wide, with a vertex that can be nearly completely smooth with two or three small nodes near the posteromedial tubercle or with slight granulation throughout the surface (Figure
The lateral surfaces are flattened and the dorsal surface is slightly convex, which gives the egg a slight bend (Figure
Measurements including the extended pinnae [mm]. Length (including operculum) 5.6–5.7, maximum width of capsule when viewed from lateral aspect 4.0–4.3 mm, length of micropylar plate 2.5–2.6 mm.
General color throughout the body (including head and antennae) is dark brown to black (Figure
Patronym, named after Garda Bagus (Java, Indonesia) who has helped to collect and rear several Phyllium species over the years and who has been instrumental in getting these established in the phasmid breeding community.
The distribution is indicated by (S) for Sumatra; (J) for Java; or (S, J) for both islands.
Phyllium (Pulchriphyllium) giganteum Hausleithner, 1984 (S, J)
Phyllium (Pulchriphyllium) pulchrifolium Audinet-Serville, 1838 (S, J)
= magdelainei Lucas, 1857
Phyllium (Pulchriphyllium) bioculatum Gray, 1832 (S)
Phyllium (Pulchriphyllium) abdulfatahi Seow-Choen, 2017 (S)
Phyllium (Pulchriphyllium) shurei Cumming and Le Tirant, 2018 (J)
Phyllium (Phyllium) jacobsoni Rehn & Rehn, 1934 (J, Sumatran record presented in
Phyllium (Phyllium) nisus sp. nov. (S) (holotype Figure
Phyllium (Phyllium) gardabagusi sp. nov. (J) (holotype Figure
Phyllium (Phyllium) hausleithneri Brock, 1999. Only known from Peninsular Malaysia; the population from Sumatra discussed in
Phyllium (Pulchriphyllium) mannani Seow-Choen, 2017. This species was erroneously recorded from Sumatra based on a male Phyllium bioculatum Gray, 1832 specimen from the
1 Antennae length, P. mannani = 18–21 mm (shorter or same length as tegmina when at rest along the dorsal surface), P. bioculatum = 26–28 mm (antennae always longer than tegmina).
2 Tegmina length, P. mannani = 14–15.5 mm, P. bioculatum = 11–11.5 mm.
3 Widest abdominal segment, P. mannani = segment VI, P. bioculatum = segment V.
4 Protibial exterior lobe, P. mannani = nearly absent, only a slight sliver, P. bioculatum = distal end notably wider than the remainder of the length.
5 Mesofemoral exterior lobe shape, P. mannani = V-shaped, P. bioculatum = U-shaped.
To date no records of Phyllium mannani from Sumatra are known to exist, and based on the above morphological characters, the specimen in the
Our molecular results reveal that the two Javan species are the sister group of the Sumatran and Peninsular Malaysian sister species, Phyllium nisus and Phyllium hausleithneri, respectively. Although we were able to examine an extensive range of specimens for all three species with purple coxae, their wide range of morphological variation has not allowed reliable external morphological differentiation. There is however the possibility of internal genitalia structures which might allow morphological differentiation, but despite dissection of several individuals no such structures could be identified. With all purple coxae species morphologically inseparable as adults, only the eggs of P. nisus and P. hausleithneri, can be distinguishable by their coloration (dark brown in P. hausleithneri Figure
The significance of wing venation traits for Phylliidae systematics has been emphasized before (
The purple coxae coloration observed in P. hausleithneri and the two newly described species P. gardabagusi and P. nisus is likely a homologous trait between these three taxa, given their close relationship as recovered here (Figure
The present study is a first step towards understanding the phylogeny, taxonomy, historical biogeography, and evolution of leaf insects. Preliminary molecular data indicated that the leaf insect genera Phyllium and Chitoniscus both do not form monophyletic groups (
The authors thank René Limoges (Canada), entomological technician at the Montreal Insectarium, Canada, for taking many photos for this work, as well as for many professional courtesies. We thank Bruno Kneubühler (Switzerland) for sharing so many amazing photographs of living individuals of these beautiful species and allowing us to use them in our publication. We thank Tim Van molle (Belgium) for allowing us to use his photograph of the three purple coxae species side by side for Figure
Table S1. GenBank accession numbers for 55 sequences from Phyllium specimens, including sex, type designation, specimen collection code, and collection data
Table S2. Primers and corresponding information for genes used in this study
Table S3. Measurements of Phyllium nisus sp. nov., holotype female, para- type males and females
Table S4. Measurements of Phyllium gardabagusi sp. nov., holotype female, paratype males and females
Explanation note: All measurements made to the nearest 0.1 mm. Measurements for paratypes are given with a minimum to maximum range. *Including cerci and head, excluding antennae. **Only one female specimen [Coll RC 16-203] had the alae exposed to measure.
File S1. Concatenated supermatrix of COI, 28S, and 16S sequences from the 21 specimens sampled (18 Phyllium and three outgroups)
Explanation note: Supermatrix of 16 COI, 18 28S, and 21 16S sequences from the 21 specimens sampled (18 Phyllium and three outgroups).
File S2. Original newick-formatted tree file inferred from COI, 28S, and 16S using IQ-TREE
Explanation note: Original tree file based on 16 COI, 18 28S, and 21 16S sequences.
File S3. Deposition of paratype material for Phyllium (Phyllium) gardabagusi sp. nov. and Phyllium (Phyllium) nisus sp. nov.