Characterization of Mitochondrial Genomes of Three Andrena Bees (Apoidea: Andrenidae) and Insights Into the Phylogenetics

International Journal of Biological Macromolecules 127 (2019) 118–125

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Characterization of mitochondrial genomes of three Andrena bees (Apoidea: Andrenidae) and insights into the phylogenetics

Bo He a,b,1, Tianjuan Su c,1,ZeqingNiuc,ZeyangZhoua, Zhanying Gu b,⁎, Dunyuan Huang a,⁎ a Chongqing Key Laboratory of Vector Insects, Chongqing Key Laboratory of Animal Biology, Chongqing Normal University, Chongqing 401331, China b Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees of Ministry of Education, Key Laboratory of Non-Wood Forest Products of State Forestry Administration, Central South University of Forestry and Technology, Changsha 410004, China c Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China article info abstract

Article history: Andrena is a large bee genus of N1500 species, which includes many important pollinators of agricultural systems. Received 2 October 2018 In this study, we present three mitochondrial genomes (mitogenomes) of Andrena species, which are the polli- Received in revised form 8 January 2019 nators of Camellia oleifera. Compared with putative ancestral gene arrangement of insects, the three Accepted 8 January 2019 mitogenomes present identical gene rearrangement events, including local inversion (trnR) and gene shufﬂing Available online 09 January 2019 (trnQ/trnM, trnK/trnD, and trnW/trnC-trnY). Most PCGs initiate with standard ATN codon and share the stop codon of TAA or TAG, whereas truncated stop codon T was detected in the atp6 gene of A. chekiangensis.Further- Keywords: Andrena more, the nad4 gene end with a single T in all three Andrena species. All tRNAs could be folded into clover-leaf Phylogeny secondary structure except for trnS1, with the dihydrouracil (DHU) arm forming a simple loop. Phylogenetic Mitochondrial genome analysis is performed on 17 Andrena mitogenomes. Maximum likelihood and Bayesian methods generate identical topology, in which A. hunanensis and A. striata form a group and are close to A. camellia. Although A. chekiangensis is also difﬁcult to be distinguished from A. camellia by morphological methods, A. chekiangensis and A. haemorrhoa form a clade and are grouped with the other taxa of the genus Andrena. © 2019 Published by Elsevier B.V.

1. Introduction Andrena Fabricius is a large bee genus of N1500 species, which is distributed predominantly in Holarctic [10,11]. These bees are important Camellia oleifera Abel. is the most important woody oil tree in China, pollinators of agricultural systems and nest solitarily or communally in with the cultivated area of approximately 3.7 million hectares [1]. It is the ground [12]. While most Andrena species fly in the spring, there self-sterile because of prezygotic late-acting self-incompatibility [2,3], are also bivoltine and univoltine taxa that emerge in summer and au- with sexual reproduction dependent on the pollinators [4]. C. oleifera tumn. They exhibit a range of diet breadth, from polylectic to oligolectic blooms from autumn to winter, during which the pollinators are limited [13,14]. Therefore, the genus Andrena is an excellent group to study the due to the low temperature. It has been reported that Andrena camellia evolution of pollen diet [15,16]. However, although Andrena species (Andrenidae) and Colletes gigas (Colletidae) are the main pollinators of have a wide range of biological characteristics, they are so morphologi- C. oleifera [4–8]. Although our field observations find that Andrena cally uniform to be distinguished from each other. It would also be a chekiangensis, Andrena hunanensis,andAndrena striata are also widely challenge to clarify their phylogenetic relationships. distributed in oil-tea forest, they are rarely described as the pollinators. The typical insect mitochondrial genome (mitogenome) is a The reason may be that these three flower visitors exhibit similar mor- 15–18 kb circular molecule, including 13 protein-coding genes (PCGs), phologic features with A. camellia [9]. They may be incorrectly identified two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), as A. camellia. Therefore, it is important to clarify the phylogenetic rela- and a control region that contains the initial sites of replication and tran- tionships of the pollinators of C. oleifera. scription [17–19]. Owing to some unique characters like small size, ma- ternal inheritance, strict orthologous genes, fast evolution rate, and low rate of recombination, mitogenome has been widely used as a molecular marker for comparative and evolutionary genomics, and phyloge- ⁎ Corresponding authors. netic analysis at different taxonomic levels [19–21]. E-mail addresses: [email protected] (Z. Gu), [email protected] fi (D. Huang). To date, fteen mitogenomes of Andrena are available in GenBank 1 These authors contributed equally to this work. [22,23], which would increase our understanding about taxonomic

Table 1 2. Materials and methods Summary of mitogenomes used in this study.

Family Species Total size (bp) Genbank accession no. 2.1. Samples collection and DNA extraction

Andrenidae Andrena angustior 15,252 KT164658 Andrenidae Andrena bicolor 15,422 KT164666 Three Andrena species (A. chekiangensis, A. hunanensis,andA. striata) Andrenidae Andrena chrysosceles 15,692 KT164602 were collected from oil-tea forest of Jiangxi, China, in November 2017. Andrenidae Andrena cineraria 17,069 KT164628 All specimens were stored in absolute ethyl alcohol at −20 °C freezer Andrenidae Andrena dorsata 16,333 KT164633 in Key Laboratory of Animal Biology, Chongqing Normal University. Andrenidae Andrena haemorrhoa 15,936 KT164645, KT164635 Andrenidae Andrena semilaevis 16,459 KT164629 Total genomic DNA was extracted separately from each specimen Andrenidae Andrena minutula 15,302 KT164675 with the Tissure DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following Andrenidae Andrena subopaca 14,747 KT164612 the manufacturer's instructions. Andrenidae Andrena flavipes 15,074 KT164679 Andrenidae Andrena fulva 15,318 KT164623 2.2. Sequencing and assembly Andrenidae Andrena labiata 15,074 KT164613 Andrenidae Andrena nigroaenea 15,376 KT164665 Andrenidae Andrena nitida 14,996 KT164636 The mitogenomes were generated by next-generation sequencing. Andrenidae Andrena camellia 15,065 KX241615 After the exacted total genomic DNA was quantified, sequences were Andrenidae Andrena chekiangensis 15,804 MH982580 fragmented to an average size of 450 bp using Covaris M220 system. Andrenidae Andrena hunanensis 14,780 MH982581 Andrenidae Andrena striata 14,736 MH982582 The library with two indexes was constructed using the Illumina Halictidae Seladonia tumulorum 15,268 KT164609 TruSeq™ DNA Sample Prep Kit (Illumina, San Diego, CA, USA) and se- Colletidae Colletes gigas 15,885 KM978210 quenced by the platform Illumina Hiseq 4000 with the strategy of 360 Colletidae Hylaeus dilatatus 15,475 NC_026468 paired-ends. Approximately 10 Gb paired-end reads of 150 bp length were generated. The quality of raw sequences was assessed using and phylogenetic relationships of this genus. In this study, we se- FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). quenced three other mitogenomes of Andrena species, compared the The software of FASTX toolkit 0.013 (http://hannonlab.cshl.edu/fastx_ characters in detail and analyzed their phylogenetic relationships. toolkit/)wasalsousedtofilter the low quality sequences. The

Fig. 1. Circular map of the three Andrena mitogenomes. Different color exhibits the nucleotide identity of BLAST searches. The species from outside to inside as follows, respectively: A. chekiangensis, A. striata,andA. hunanensis. 120 B. He et al. / International Journal of Biological Macromolecules 127 (2019) 118–125

Andrena angustior Andrena flavipes 0 Andrena dorsata Andrena semilaevis Andrena minutula Andrena subopaca 1.12 Andrena nigroaenea Andrena nitida

Andrena camellia 2.25 Andrena chekiangensis Andrena fulva Andrena hunanensis 3.38 Andrena striata Andrena haemorrhoa Andrena labiata 4.5 Andrena bicolor Andrena cineraria TGT(C) GTC(V) CTC(L) ACG(T) CCG(P) CAG(Q) CAC(H) AGT(S) CAT(H) ACT(T) GTT(V) CGA(R) GCA(A) TCT(S) CAA(Q) CCC(P) GCC(A) TCC(S) GAC(D) ATC(I) GAG(E) CTT(L) ACC(T) TAC(Y) AAC(N) TTC(F) AAG(K) ATG(M) TGG(W) AGG(S) CGC(R) AGC(S) CTG(L) TCG(S) GGC(G) GCG(A) CGG(R) GTG(V) TGC(C) GGG(G) TTG(L) CTA(L) CGT(R) GGT(G) CCT(P) TTA(L) TCA(S) ACA(T) CCA(P) TGA(W) AGA(S) GTA(V) TAT(Y) TTT(F) AAA(K) ATT(I) GAA(E) GGA(G) ATA(M) AAT(N) GAT(D) GCT(A)

Fig. 2. The relative synonymous codon usage (RSCU) of PCGs in Andrena mitogenomes. The x-axis and y-axis indicate the hierarchical clustering of codon frequencies and Andrena species, respectively.

downstream analyses were performed on clean data of high quality substitution models were confirmed by PartitionFinder 2.1.1 [37]with (Q20 N 90% and Q30 N 85%). The mitogenomes were reconstructed by the Bayesian Information Criterion (BIC). The sequences were pre- MITObim v1.7 [24] with the default parameters, and the mitogenome defined by both gene types (13 PCGs, 22 tRNAs, and two rRNAs) and of A. camellia (GenBank accession no: KX241615) was employed as a codon positions (the first, second, and third codon positions of each reference. PCG). The maximum likelihood (ML) analysis was inferred using IQ-TREE 2.3. Bioinformatic analysis [38]. Branch support was conducted with 1000 replicates of ultrafast likelihood bootstrap. Bayesian inference (BI) analysis was estimated The secondary structures of tRNAs were predicted by Mitos using MrBayes 3.2.6 [39]. Two independent runs were performed, WebServer [25] under the invertebrate mitochondrial genetic code. each with three hot chains and one cold chain, with posterior distribu- The PCGs and rRNAs boundaries were determined by the positions of tions estimated using Markov Chain Monte Carlo (MCMC) sampling. tRNAs, and by alignment with other Andrena gene sequences. In addi- The MCMC chains were set for 10,000,000 generations, with tree sam- tion, to ensure more accurate gene boundaries, PCGs were also trans- pling every 1000 steps and a relative burn-in of 25%. The convergence lated into amino acids. The comparable sequence identity map was of the two runs was evaluated by average standard deviation of split fre- generated by CGView Comparison Tool [26]. Secondary structures of quencies (b0.01). The phylogenetic trees were drawn by FigTree 1.4.2 both rrnL and rrnS were inferred following the models proposed for (http://tree.bio.ed.ac.uk/software/figtree/). some other hymenopterans [27–30]. Helix numbering was described according to the convention of Comparative RNA Web (CRW) Site 3. Results and discussion [31]. In addition, Mfold Web Server was also used to fold the sequences that lack significant homology [32]. The base composition, codon 3.1. Genome structure usage, and Relative synonymous codon usage (RSCU) were calculated by MEGA 6.05 [33]. The AT-skew and GC-skew were computed accord- The mitogenomes of A. chekiangensis (GenBank accession number ing to the following formulas: AT-skew = [A − T] / [A + T] and GC- MH982580), A. hunanensis (MH982581), and A. striata (MH982582) skew = [G − C] / [G + C] [34]. were sequenced, with the length of 15,804 bp, 14,780 bp, and 14,736 bp, respectively (Table 1). Due to high A + T content and compli- 2.4. Phylogeny analysis cated secondary structure, the control regions of A. hunanensis and A. striata were unable to be amplified. The number of reads mapping Phylogenetic analyses were performed on 17 Andrena species, with for the A. chekiangensis, A. hunanensis, and A. striata was 69,290, outgroups from the families of Halictidae and Colletidae (Table 1). Nu- 122,074, and 109,497, respectively. The sequence coverage for all cleotide sequences for each of the 13 PCGs were translated into amino the three mitogenomes was 100%. The sequence depth for the acids, aligned separately with Muscle implemented within MEGA 6.05, mitogenomes of A. chekiangensis, A. hunanensis, and A. striata was and then toggled back into nucleotide alignments. The tRNAs and 641×, 1083×, and 1028×, respectively. Each mitogenome contained rRNAs were aligned using MAFFT 7.310 with the Q-INS-i algorithm the entire set of 37 genes, most of which located on the J-strand (9 [35]. In addition, Gblock 0.91b [36] was used to eliminate unreliably PCGs and 13 tRNAs). The remaining genes (4 PCGs, two rRNAs, and 9 aligned sequences. The best-fit partitioning schemes and nucleotide tRNAs) were coded on the N-strand (Fig. 1; Table S1). Compared with B. He et al. / International Journal of Biological Macromolecules 127 (2019) 118–125 121

Fig. 3. Secondary structures of 22 tRNAs in the mitogenome of A. chekiangensis. Filled circle, nucleotide conserved in Andrena mitogenomes; hollowed circle, nucleotide not conserved. Watson-Crick base pairings, G-U bonds, and mismatches are represented by dashes, solid dots, and hollowed dots, respectively.

putative ancestral gene arrangement of insects [18,19], the three 3.2. Nucleotide composition mitogenomes presented identical gene rearrangements, including local inversion (trnR)andgeneshufﬂing (trnQ/trnM, trnK/trnD,and The nucleotide composition of A. chekiangensis (77.92%), trnW/trnC-trnY), which were also consistent with other mitogenomes A. hunanensis (75.95%), and A. striata (75.86%) was biased toward A reported in the same genus of Andrena [23]. and T (Table S2). This bias fell within the range observed in the previ- To better visualize the gene identity in the three newly sequenced ously sequenced mitogenomes of Andrena species, from 74.09% in Andrena mitogenomes, the comparable circular map was generated Andrena ﬂavipes to 80.27% in Andrena angustior. The A+T content of (Fig. 1). Among different regions, high conservation was observed in PCGs, tRNAs, and rRNAs of the three mitogenomes also fell within the rrnL, rrnS, and some tRNAs (e.g., trnM, trnG,andtrnV). Among PCGs, cy- range observed for the sequenced Andrena species. In addition, AT- tochrome oxidase genes (e.g. cox1 and cox2) and the cytochrome b gene skews and GC-skews in the newly sequenced mitogenomes were simi- (cob) were more conserved, whereas NADH dehydrogenase subunit lar to patterns found in other Andrena species, i.e., more A and C than T genes were more variable (e.g. nad3, nad4l,andnad6). and G in the majority strand [23]. Comparative analyses of other 122 B. He et al. / International Journal of Biological Macromolecules 127 (2019) 118–125

Fig. 4. Predicted secondary structure of the rrnL in the mitogenome of A. chekiangensis. Filled circle, nucleotide conserved in Andrena mitogenomes; hollowed circle, nucleotide not conserved. Roman numerals indicate the conserved domains. Watson-Crick pairs are illustrated by dashes, whereas G-U pairs are joined by dots.

hymenopteran species also showed that most of the AT-skews were stem (5 bp), and acceptor stem (7 bp) were conserved in length. Except positive, while most GC-skews were negative [28]. for trnS1, the length of DHU and TΨC stems was 3–4bpand3–5 bp, respectively. However, The DHU and TΨCloopsandtheextraarm 3.3. Protein-coding genes were more variable, with obvious length variation and nucleotide substitutions. In addition, unmatched base pairs were scattered throughout The three newly sequenced Andrena mitogenomes exhibited similar the tRNA stems, including noncanonical match of G-U and mismatches start and stop codons (Table S1). All the PCGs initiated with the typical of A-A, A-C, U-C, and U-U. These wobble and mismatched pairs had also ATN codon. While most PCGs ended with TAA or TAG, truncated been proposed in other hymenopteran mitogenomes [27–30]. They stop codon T was also detected in the atp6 gene of A. chekiangensis. might be restored by the post-transcriptional editing processes [41]or Furthermore, the nad4 gene end with a single T in all three Andrena represented unusual pairings [31]. species. Truncated stop codons could be commonly found in insect mitogenomes and were thought to be complemented by post- 3.5. Ribosomal RNA genes transcriptional polyadenylation [40]. Relative synonymous codon usage indicated that A/T was more fre- The secondary structure of rrnL consisted of ﬁve domains (domain III quently used than G/C in degenerate codons (Fig. 2). For example, the was absent in insects) and 44 helices. The multiple alignments of third codon positions of the six most frequently used codons in Andrena, Andrena rrnL spanned 1322 positions and contained 786 conserved includingTTA,TCA,GTT,ACA,CCA,andCGA,wereallcomposedofAor (59.46%) and 536 variable sites (40.54%), respectively. Conserved nucle- T. Conversely, some GC-rich codons were seldom used in Andrena spe- otides were distributed unevenly, with domains IV and V more con- cies, such as CGC, GGC, CCG, and GCG. served than other domains (Fig. 4). Within domain IV, four helices (H1775, H1830, H1906, and H1952) were highly conserved, with only 3.4. Transfer RNA genes 0–1 nucleotide substitutions. In domain V, most helices were conserved, except for helices H2077 and H2347. In the variable domains I, II, and VI, All 22 tRNAs typical of arthropod mitogenomes were found in the except for the helices H563 and H822, there were no obvious conserved three Andrena mitogenomes (Fig. 3). Most tRNAs could be folded into helices. clover-leaf secondary structure except for trnS1, with the dihydrouracil The rrnS contained 28 helices in three domains. The multiple align- (DHU) arm forming a simple loop. The anticodon loop (7 bp), anticodon ments of Andrena rrnS spanned 768 positions and contained 452 B. He et al. / International Journal of Biological Macromolecules 127 (2019) 118–125 123

Fig. 5. Predicted secondary structure of the rrnS in the mitogenome of A. chekiangensis. Filled circle, nucleotide conserved in Andrena mitogenomes; hollowed circle, nucleotide not conserved. Roman numerals indicate the conserved domains. Watson-Crick pairs are illustrated by dashes, whereas G-U pairs are joined by dots.

conserved (58.85%) and 316 variable positions (41.15%), respectively. many species of the genus Andrena [23]. Of the three newly Nucleotide conservation was also distributed unevenly (Fig. 5). Com- sequenced species in the present study, A. hunanensis and pared to domains I and II, domain III was more conserved within A. striata formed a group and were close to A. camellia, with the Andrena, except for the helices H1068, H1074, and H1113. In domain I, posterior probabilities of 1.0 and ML bootstraps of 100. Although the helix H47 was highly variable. To date, no consistent secondary A. chekiangensis was also difﬁcult to be distinguished from structure had been proposed for this region in insects [42]. In domain A. camellia by traditional morphological methods, A. chekiangensis II, although helix H673 was reported to form various patterns in and A. haemorrhoa formed a clade and were grouped with the other mitogenomes of insects [43–45], it formed a relatively conserved struc- taxa of the genus Andrena. ture in hymenopterans [27–30,46]. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.01.036. 3.6. Phylogenetic analysis Declarations of interest Phylogenetic analyses were performed on concatenated nucleotide sequences of 13 PCGs, two rRNAs, and 22 tRNAs derived from The authors declare no conﬂict of interest. 17 Andrena mitogenomes, with one Halictidae and two Colletidae speciesastheoutgroups[23,47–49]. The BI and ML analyses gener- Acknowledgements ated identical topology structure (Fig. 6). Except for the three newly sequenced species, the phylogenetic relationships among This work was supported by the National Natural Science Founda- the Andrena species also supported the conclusion from a recent tion of Chongqing [grant number cstc2018jcyjAX0382]; and the Na- study about phylogenetic relationships of bees, which also include tional Natural Science Foundation of China [grant number 31770160]. 124 B. He et al. / International Journal of Biological Macromolecules 127 (2019) 118–125

Fig. 6. Phylogenetic tree inferred from mitogenomes of Andrena. Numbers at the nodes are Bayesian posterior probabilities and ML bootstrap values.

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