DNA barcoding reveals polymorphism in the pygmy grasshopper Tetrix bolivari (Orthoptera, Tetrigidae)

Ling Zhao; Li-Liang Ling; Zhe-Min Zheng

doi:10.3897/zookeys.582.6301

Abstract

Many pygmy grasshopper species exhibit colour-marking polymorphism. However, this polymorphism in some species, such as Tetrix bolivari, is almost unknown. The aim of this work is to identify using DNA barcoding the colour-marking polymorphic morphs of this pygmy grasshopper species collected from both grass and sand microhabitats. Analysis by NJ clustering and pairwise distances indicated that all specimens collected showing colour-marking polymorphism are species of T. bolivari. Haplotype network construction showed ten different haplotypes from a total of 57 T. bolivari individuals with H1(82.5%) being the most common type and it also displayed low divergence within T. bolivari population. The haplotype analyses were consistent with the NJ clustering. Our field census showed the frequency of T. bolivari morphs differed significantly, with the rank order of morphs (from high to low) typeA₁, type B₁, type A₂, type A₃, type A₄, type A₅, type A₆, type A₇, type B₂, type B₃, and type B₄. The most common type A morphs were without contrasting markings, while the rarer type B morphs have contrasting white markings. We suggest that type B morphs have greater camouflage effects against natural backgrounds such as grass or sand than type A morphs. Both our field census and haplotype analysis revealed that type A has higher frequency and more haplotypes than type B.

Keywords

Crypsis, DNA barcoding, frequency, polymorphism, Tetrix bolivari

Introduction

Pygmy grasshoppers are typical examples of polymorphic species (Holst 1986, Ichikawa et al. 2006). Different species show colour-marking polymorphism. Moreover, some species are highly polymorphic in colour and markings even within a single population (Forsman 2000). Such polymorphism has adaptive significance which can provide camouflage for the species against their natural backgrounds (crypsis), such as grass or sand (Ruxton et al. 2004, Stevens and Merilaita 2009, Cott 1940, Thayer 1909). The degree of camouflage differed among the colour-marking polymorphic morphs. Usually morphs with contrasting markings have greater camouflage against their natural backgrounds than those without them. However, the degree of camouflage of the morphs was not consistent with the morph frequency. It was reported the frequency of morphs with various types of markings differed significantly between the grass and sand microhabitats. Overall, morphs with contrasting markings were rarer in both microhabitats, although they were more cryptic (Kaori et al. 2010). The more cryptic morphs are not common in the microhabitats. Furthermore, for certain morphs with contrasting marking, such as longitudinal morphs of Tetrix japonica, they tended to be more common in the sand microhabitat where they were more conspicuous compared to the grass background where the markings provided a stronger camouflage effect for them. Therefore, the morph frequency cannot reflect the degree of crypsis.

Many pygmy grasshopper species exhibit discontinuous variation in colour and pattern of the pronotum, such as Tetrix japonica (Kaori et al. 2010), Tetrix undulate (Ahnesjo and Forsman 2003) and Tetrix subulata (Forsman 1997, 2000, 2001) and there is a strong tendency for the general patterns to be repeated in different genera and species (Nabours 1929, Fisher 1939). However, there are no publications on polymorphism in pygmy grasshoppers in China.

The Tetrigidae is an ancient group of Orthoptera with relatively uniform body structure. Most Tetrigidae are small, inconspicuous orthopterans about one cm long. They are terricolous and inhabit humid habitats, and some species are semi-aquatic (Podgornaya 1983, Paranjape et al. 1987).

In China, the Family Tetrigidae contains 15 genera, including the large genus Tetrix, which currently has 88 species (Deng et al. 2008). Generally, this Family of grasshoppers is among the least-studied groups of Orthoptera and most publications mainly focused on descriptions of new species and morphological taxonomy in China. Their ecology and biology are almost unknown.

Tetrix bolivari is one of the least studied species of China Tetrigidae but with wide distribution. Few published reports provide molecular data about its phylogeny (Fang et al. 2010, Chen and Jiang 2004, Jiang et al. 2002), and data on the activity, feeding biology and vibratory communication can be found in one study (Petr et al. 2011). Polymorphism within T. bolivari adults has not been reported.

In this paper, we collected pygmy grasshoppers from both grass and sand microhabitats with large variation in body colouration and markings. To examine whether they are the same species with various colour-marking morphs or they are the different species, we conducted identification experiments using the protein-coding cytochrome c oxidase subunit I (COI) region as a DNA barcode. In addition, we conducted a field census of the morphs in the microhabitats (sand and grass) to confirm the grasshopper morph frequency.

Materials and methods

Sampling

Adult pygmy grasshoppers were collected from both grass and sand microhabitats in Mianyang, Sichuan Province, China in July to August, 2013. All morphs are characterized by both a long pronotum that extends beyond the apex of the abdomen and highly reduced forewings. They are small (males, 11.8–16.0 mm; females, 13.5–17.0 mm) and exhibit extraordinary variation in the colour and markings of the pronotum from black, through yellowish-brown to light grey or white, with some individuals being monochrome and others having spots, markings or distinct patterns on the pronotum (and also on the hind legs) such as a narrow light yellowish longitudinal stripe on the mid-line of the upper surface of the pronotum or whitish and blackish markings on the dorsal surface of the pronotum.

Fifty-seven specimens of different colour-marking morphs were preserved in 100% ethanol and stored at –4 °C for identification experiments.

Identification experiments

DNA extraction: DNA from the tissues of the grasshoppers was extracted from the hind leg using a routine phenol/chloroform method (Zhou et al. 2007).

PCR amplification and sequencing: The DNA was ampliﬁed using polymerase chain reaction (PCR) in an ABI thermocycler. The following primers were used for amplication of the COI gene: 5’-TYTCAACAAAYCAYAARGATATTGG-3’ and 5’-T AAACTTCWGGRTGWCCAAARAA TCA-3’. PCR reaction was carried out in a total volume of 15 ul containing 1ul DNA template, 7.5 ul Mix (2×Taq DNA Polymerase, 2×PCR Buffer, 2×dNTP), 1 ul of each primer and 4.5 ul PCR-grade RNase-free water. Thermo-cycling conditions were as follows: one initial cycle of 4 min at 94 °C followed by 35 cycles of 94 °C for 15 s, 46 °C for 20 s, 70 °C for 90 s, with ﬁnal step of 72 °C for 7 min. The PCR products were visualized on 1% agarose gels and then sequenced with both the forward and reverse primers by Shanghai SANGON after separation and purification.

Data analysis: The 57 sequences were aligned using CLUSTALX and the standard 658bp was kept for the following analysis (GenBank Accession nos KU570134-KU570190). The morphological identiﬁcation for one individual (No. 57) suggested it was Tetrix bolivari (Tetrigidae, Tetriginiae, Tetrix). The 56 remaining individuals plus the individual T. bolivari were analyzed together in NJ analysis by MEGA version 5.0, together with another 4 Tetrix species, 1 Alulatettix yunnanensis and Teleogryllus emma as outgroup.

The Kimura 2-parameter (K2P) model of base substitution was used to calculate pairwise genetic distance in MEGA 5 software. Species discrimination in DNA barcoding studies also depends on establishment of threshold interspecies nucleotide divergence. In this study, nucleotide divergence of 3% was considered as a threshold between species as observed in Orthopterans (Mao 2011).

The haplotype network based on 658 base pairs of COI sequences was constructed using the median-joining algorithm (Bandelt et al. 1999) implemented in NETWORK 4.1 (Forster et al. 2000).

Field survey of the frequency of grasshopper morphs

Grasshoppers were collected using random sweeps with an insect net in both microhabitats and we counted the different colour-marking morphs in the field. Only adults were used in this research.

Results

Identification by DNA barcoding

Our NJ analysis showed that the 56 individuals with different colour-marking morphs clustered with T. bolivari into one clade with bootstrap value of 100% (Fig. 1). The pairwise distances indicated the nucleotide divergence varied from 0.0% to 0.6% and the overall mean distance was 0.1%, which was far less than the threshold of 3% used for species discrimination (Suppl. material 1). According to the above analysis, we inferred that all pygmy grasshoppers with different morph types in this research are all species of T. bolivari. Meanwhile, the NJ analysis showed that T. bolivari, T. qinlingensis, Alulatettix yunnanensis and T. japonica have close relationship with a bootstrap value of 100%. However, another two species T. ornata and T. brunnerii were separated from the other species by interspecies nucleotide divergence ranging from 16.1% to 18%.

Figure 1.

NJ clustering analysis of 62 sequences of pygmy grasshoppers using MEGA5 and K2P model. Teleogryllus emma is used as an outgroup. The clade 1-57 represents samples from this study.

Haplotype network construction

The haplotype analyses based on the 62 sequences were consistent with the NJ clustering (Fig. 2A). Ten different haplotypes were found in T. bolivari population (Fig. 2). The most frequent haplotype was H1, occurring in 82.5% of individuals sampled (Table 1), indicating a low degree of variation in this population. This type was followed by H4 with much lower frequency (3.5%) and the remaining haplotypes occurred at a frequency of 1.8% (Table 1). Haplotypes H3, H4, H5, H6, H8 and H9 were each linked to H1 by a single nucleotide substitution at positions 3, 394, 343, 355, 28 and 235, respectively (Fig. 2A). The haplotype H7 was the most distant haplotype. It was separated from the haplotype H1 by 3 mutational events (Fig. 2).

Figure 2.

A Haplotype network from 63 sequences, including 57 Tetrix bolivari individuals, 4 Tetrix species, 1 Alulatettix yunnanensis and 1 outgroup (Teleogryllus emma) B Haplotype network of 57 Tetrix bolivari individuals combined with the morph types. Circle size is proportional to haplotype frequency. Lines drawn between haplotypes represent mutation events identified by the numbers corresponding to the positions at which the mutations were observed. Red points represent hypothetical haplotypes (median vector). Colours in B represent morph types. Yellow areas represent type A and black areas represent type B.

Table 1.

Download as

CSV

XLSX

Frequency of the ten different haplotypes based on the 658 bp COI region in 57 Tetrix bolivari individuals used in this study.

Haplotype	Sequence code	Frequency (%)	Morph type
Haplotype	Sequence code	Frequency (%)	A	B
H1	1 3 4 5 6 7 8 9 11 12 13 14 15 16 19 21 22 23 24 25 26 28 29 30 31 32 34 35 36 37 38 39 40 41 42 44 45 46 47 48 49 50 51 53 54 56 57	47 (82.5)	40	7
H2	33	1 (1.8)	1
H3	43	1 (1.8)	1
H4	2 18	2 (3.5)	2
H5	55	1 (1.8)	1
H6	10	1 (1.8)	1
H7	20	1 (1.8)	1
H8	17	1 (1.8)		1
H9	27	1 (1.8)	1
H10	52	1 (1.8)	1
Total	57		49	8

Frequency of different colour-marking morphs in the grass and sand microhabitats

A total of 343 pygmy grasshoppers (T. bolivari) were collected from both microhabitats and we categorized these morphs into 2 types (type A and type B) and 11 subtypes (type A_1-7 and type B_1-4) based on the colour and markings on the pronotum (Fig. 3). Type A generally has dark colours, such as black, brown and grey, without contrasting markings. While type B had white on the pronotum, mixed with the black markings. The number of each subtype is shown in Table 2. From this table, we can see that type A was dominant in the habitat with type A₁ more common than other subtypes (type A, 79.3%; type A₁, 29.7%), whereas type B was rare, especially type B₄ which has obvious contrasting markings (type B, 20.7%; type B₄, 0.9%).

Figure 3.

Morphs of pygmy grasshoppers (Tetrix bolivari) classified by type of colour and markings on the pronotum. A_1-7 belongs to type A; B_1-4 belongs to type B.

Table 2.

Download as

CSV

XLSX

Morph types of pygmy grasshoppers (Tetrix bolivari) classified by the colour and markings on the pronotum.

Type	Number	Percentage
A₁	102	29.7
A₂	34	9.9
A₃	31	9.0
A₄	31	9.0
A₅	28	8.2
A₆	25	7.3
A₇	21	6.1
A_1-7	272	79.3
B₁	43	12.5
B₂	15	4.4
B₃	10	2.9
B₄	3	0.9
B_1-4	71	20.7

In this study, 57 T. bolivari individuals were used in NJ clustering and haplotype analysis, including 49 type A morphs and 8 type B morphs (Table 1). These type A morphs have nine different haplotypes and the 8 type B morphs have two haplotypes (Fig. 2B). Both morph types have haplotype H1 and it is the most prevalent type in both type A and type B.

Discussion and conclusion

The aim of this study was to identify morphs of T. bolivari using DNA barcoding and examine polymorphism and morph frequency in T. bolivari. Both the NJ clustering analysis and pairwise distances indicated that all specimens in this experiment are species of T. bolivari, which exhibits polymorphism in colour-marking morphs. Furthermore, we found the non-marked morph, spotted morph, and horizontal morph in T. japonica also appeared in T. bolivari. It was reported T. bolivari is commonly found with T. subulata (Adamovic 1969, Sardet 2007). Therefore, we suspect these two species presumably share some morph types, although we didn’t find any T. subulata in our collection.

Our NJ analysis showed that Alulatettix was closely related to the three Tetrix species. Previous molecular studies (Fang et al. 2010) correspond with results of our research. From the morphological observations, Alulatettix and Tetrix belong to the subfamily Tetriginae, and both of these genera are similar in the head not projecting above upper level of pronotumand and the posterior margin of lateral lobes in lateral view with two concavities, but they differ in the shape of pronotum and degree of development of tegmina and hind wings. However, another two Tetrix species (T. ornata and T. brunnerii) were less close to the other species and showed higher interspecies nucleotide divergence, from 16.1% to 18%. Here, sequences of T. ornata and T. brunnerii downloaded from GenBank were submitted by the Biodiversity Institute of Ontario, Canada, a place far from China, while the other 2 Tetrix species (T. japonica and T. qinlingensis) and A. yunnanensis were contributed by Nanjing Normal University, China and T. bolivari was from our lab. So, we inferred that the higher nucleotide divergence between T. ornata, T. brunnerii and other pygmy grasshopper species likely reflects geographical distribution differences. In addition, the molecular phylogeny also revealed that the genus Tetrix is not monophyletic (Fang et al. 2010, Chen and Jiang 2004).

Furthermore, a total of ten different haplotypes were detected in this single colour-marking polymorphic population with H1 (82.5%) being the most common type. The haplotype network displays a shallow divergence in this T. bolivari population with a maximum of 4 base changes between the most divergent haplotypes. The haplotype analysis combined with the morph types showed type A has more haplotypes than type B and both of them have the prevalent haplotype H1.

Our field census of the polymorphism in the microhabitats (sand and grass) demonstrated that the different morphs of T. bolivari were not equivalent in the frequency, with the rank order of morphs (from high to low) being typeA₁, type B₁, type A₂, type A₃, type A₄, type A₅, type A₆, type A₇, type B₂, type B₃, type B₄. Generally, type A was more common than type B. Earlier work on T. japonica has revealed the more common morphs, usually without contrasting markings, are not more cryptic in either grass or sand microhabitat. In contrast, the more cryptic morphs which have contrasting markings were rarer in each microhabitat (Kaori et al. 2010). Our field survey also showed that the more common type A morphs usually exhibited non-marked and mono-coloured basal colouration or any number of spots on mono-coloured basal colouration, while the rarer type B morphs had contrasting white markings. So, we infer that type B morphs have a much greater degree of crypsis against the natural backgrounds, such as grass or sand than type A morphs. There is no positive association between morph frequency and the degree of crypsis, which can be explained by the differential crypsis hypothesis (Forsman 1998).

Acknowledgments

The authors are grateful to Doug Strongman for his valuable suggestions to the manuscript. This research was supported by the project of Mianyang Normal University (QD2012A05).

References

Adamovic ZR (1969) Habitat relationship of some closely related species of Tetrigidae, Orthoptera. Ekologia 4: 165–184.

Ahnesjo J, Forsman A (2003) Correlated evolution of color pattern and body size in polymorphic pygmy grasshoppers, Tetrix undulata. Journal Evolutionary Biology 16: 1308–1318. doi: 10.1046/j.1420-9101.2003.00610.x

Bandelt HJ, Forster P, RÖhl A (1999) Median-joining networks for inferring intraspecific phylogenies. Molecular Biology and Evolution 16: 37–48. doi: 10.1093/oxfordjournals.molbev.a026036

Chen AH, Jiang GF (2004) Phylogenetic relationships among 12 species of Tetrigidae (Orthoptera: Tetrigoidea) based on partial sequences of 12S and 16S ribosomal RNA. Zoological Research 25: 510–514.

Cott HB (1940) Adaptive coloration in animals. Methuen, London, 596 pp.

Deng WA, Zheng ZM, Wei SZ (2008) A new species of the genus Tetrix Latreille and a newly reported male of Bolivaritettix luochengensisDeng, Zheng et Wei, 2006 (Orthoptera: Tetrigoidea). Zoological Research 29: 455–458. doi: 10.3724/SP.J.1141.2008.00455

Fang N, Xuan WJ, Zhang YY, Jiang GF (2010) Molecular phylogeny of Chinese Tetrigoids (Orthoptera, Tetrigoidea) based on the mitochondrial cytochrome C oxidase iv gene. Acta Zootaxonomica Sinica 35: 696–702.

Fisher RA (1939) Selective forces in wild populations of Paratettix texanus. Annals of Eugenics 9: 109–122. doi: 10.1111/j.1469-1809.1939.tb02201.x

Forsman A (1997) Thermal capacity of different color morphs on the pygmy grasshopper Tetrix subulata. Annales Zoologici Fennici 34: 145–149.

Forsman A (1998) Visual predators impose correlational selection on prey color pattern and behavior. Behavioral Ecology 9: 409–413. doi: 10.1093/beheco/9.4.409

Forsman A (2000) Some like it hot: Intra-population variation in behavioral thermoregulation in color-polymorphic pygmy grasshoppers. Evolutionary Ecology 14: 25–38. doi: 10.1023/A:1011024320725

Forsman A (2001) Clutch size versus clutch interval: life history strategies in the color-polymorphic pygmy grasshopper Tetrix sublata. Oecologia 129: 357–366. doi: 10.1007/s004420100743

Forster P, Bandelt HJ, RÖhl A, Polzin T (2000) NETWORK 3.1.1.0. Software free available at: www.fluxus-engineering.com. Fluxus Technology Ltd, Cambridge.

Holst KT (1986) The Saltatoria of northern Europe. Fauna Entomologica Scandinavica 16: 1–127.

Ichikawa A, Kano Y, Kawai M, Tominago O, Murai T (2006) Orthoptera of the Japanese Archipelago in Color. Hokkaido University Press, Hokkaido, 688 pp.

Jiang GF, Lu G, Huang K, Huang RB (2002) Studies on genetic variations and phylogenetic relationships among five species of Tetrix using RAPD markers. Acta Entomologica Sinica 45: 499–502.

Kaori T, Atsushi H, Takayoshi N (2010) Camouflage effects of various colour-marking morphs against different microhabitat backgrounds in a polymorphic pygmy grasshopper Tetrix japonica. PLOS ONE 5: 1–7. doi: 10.1371/journal.pone.0011446

Mao SL (2011) A preliminary study on Acridoidea and Tettigonioidea species of China by DNA barcoding and Phylochips. PhD thesis, Shaanxi Normal University, Xi’an.

Nabours RK (1929) The genetics of the Tettigidae (Grouse Locusts). Bibliographia Genetica 5: 27–104.

Paranjape SY, Bhalerao AM, Naidu NM (1987) On etho-ecological characteristics and phylogeny of Tetrigidae. In: Bacetti BM (Ed.) Evolutionary biology of Orthopteroid insects. Ellis Horwood, New York, 386–395.

Petr K, Jaroslav H, Šárka G, David M (2011) Biology of Tetrix bolivari (Orthoptera: Tetrigidae). Central European Journal of Biology 6: 531–544.

Podgornaya LI (1983) Pryamokrylye nasyekomye semeystva Tetrigidae (Orthoptera) fauny SSSR. Trudy Zoologicheskogo Instituta AN SSSR, 112.

Ruxton GD, Sheratt TN, Speed MP (2004) Avoiding Attack: the Evolutionary Ecology of Crypsis, Warning Signals and Mimicry. Oxford University Press, Oxford, 264 pp. doi: 10.1093/acprof:oso/9780198528609.001.0001

Sardet E (2007) Tetrix bolivari Saulcy in Azam, 1901, espèce mythique ou cryptique? (Caelifera, Tetrigoidea, Tetrigidae) (Is Tetrix bolivari Saulcy in Azam, 1901, mystic or cryptic species? (Caelifera, Tetrigoidea, Tetrigidae)). Matériaux Orthoptériques et Entomocénotiques 12: 45–54. [In French]

Stevens M, Merilaita S (2009) Defining disruptive coloration and distinguishing its functions. Philosophical Transactions of the Royal Society B-Biological Sciences 364: 481–488. doi: 10.1098/rstb.2008.0216

Thayer GH (1909) Concealing-coloration in the animal kingdom: an exposition of the laws of disguise through color and pattern: being a summary of Abbott H. Thayer’s discoveries. Macmillan, New York, 469 pp. doi: 10.5962/bhl.title.57368

Zhou ZJ, Huang Y, Shi FM (2007) The mitochondrial genome of Ruspolia dubia (Orthoptera: Conocephalidae) contains a short A+T-rich region of 70bp in length. Genome 50: 855−866. doi: 10.1139/G07-057

Supplementary material

Supplementary material 1

Pairwise genetic distance (K2P) based on COI sequence using MEGA 5

Ling Zhao, Li-Liang Lin, Zhe-Min Zheng

Data type: specimens data

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.

Download file (53.00 kb)