Characterization of the complete mitochondrial genome of Hierococcyx sparverioides and phylogenetic implications

Abstract

The species Hierococcyx sparverioides belongs to the order Cuculiformes, family Cuculidae. We present the first complete mitochondrial genome of H. sparverioides (17,264 bp; GenBank PX733929), comprising 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a control region, with a structure similar to other Cuculidae. We conducted mitogenome assembly verification, selective pressure and codon usage (RSCU) analyses, tRNA secondary structure predictions, and phylogenetic reconstruction. This study provides novel mitogenome data contributing to understanding of the evolution and phylogenetic relationships of Hierococcyx species.

Introduction

The Cuculidae family is an ancient and diverse group comprising approximately 150 species worldwide (Dos Santos et al. 2020; Kretschmer et al. 2024). The family Cuculidae has been a subject of significant interest in the fields of evolutionary and ecological studies. This interest stems from their distinctive brood-parasitic nesting strategy, complex vocalization systems, and broad geographic distribution. Hierococcyx sparverioides (Vigors, 1832) is classified within the order Cuculiformes and the family Cuculidae. H. sparverioides, a species of parasitic cuckoo, has been the focus of numerous studies examining vocalization rhythms or host use (Yang et al. 2012, 2015; Yi et al. 2020). However, several key questions regarding the evolutionary history and phylogenetic relationships of H. sparverioides remain unresolved. The phylogenetic relationship has been inferred from only a few mitochondrial fragments (e.g. COX I (Fossøy et al. 2016) and ND2 (Wang et al. 2025)), and whether this placement is robust when richer genomic data are used has not been tested. Phylogenetic signal from individual genes is often limited and can be misleading due to factors such as mutational saturation or stochastic noise, which may lead to incongruent or poorly supported topologies (Cummings et al. 1995; Lozano-Fernandez 2022). Additionally, parasitic cuckoos exhibit unusual life-history strategies, including hawk mimicry by adults (Welbergen and Davies 2011) and visual mimicry of host nestlings (Langmore et al. 2011), yet whether these strategies are associated with distinct selective pressures on mitochondrial genes relative to non-parasitic lineages remains unclear. A complete mitogenome can offer a more robust dataset and thus help test whether parasitic lineages show different selection pressures (dN/dS) compared to non-parasitic ones. Although previous studies have included H. sparverioides using individual gene fragments, its complete mitochondrial genome has not yet been reported. Mitochondrial genome data are widely used in studies of molecular evolution and taxonomic clarification (Kim et al. 2026). In comparison with analyses based on a poorly resolved set of fragments, complete mitochondrial genomes provide substantially richer genetic information and yield clearer and more robust phylogenetic inferences. Therefore, we characterized the complete mitochondrial genome of H. sparverioides using high-throughput sequencing and inferred its phylogenetic relationships based on concatenated mitochondrial protein-coding genes (PCGs). This enhances our understanding of the evolutionary history of the species under investigation and will provide valuable mitochondrial genomic resources to support future phylogenetic studies of birds in the order Cuculiformes, with a particular focus on parasitic lineages. Specifically, the complete mitogenome of H. sparverioides enables testing of whether Hierococcyx is indeed sister to Cuculus, whether the genus merits independent taxonomic status.

Materials and methods

Given that mitochondrial genomes are generally highly conserved within a species and the objective of this study is to characterize the mitogenome structure rather than to assess intraspecific variation, a single specimen is sufficient for this purpose. A specimen of H. sparverioides was collected on October 13, 2025, from the Jinshanyakou bird banding site, Gasa Town, Xinping County, Yuxi City, Yunnan Province, China (latitude: 23°48’20.80ʺN; longitude: 101°35′58.85ʺE) (Figure 1). Prior to blood sampling, the species identification was confirmed by Dr. Heqi Wu, a leading expert in the field. A small volume of blood was collected by puncturing the wing vein with a sterile disposable lancet of the type used for blood glucose testing, and the blood was then absorbed onto sterile cotton swabs. Two cotton swabs were collected from the specimen—one for genomic DNA extraction and one preserved as a backup. The blood samples (voucher ID: 130B_XP_DYJ_1012_XY) were stored in ethanol at −20 °C at the Kunming Institute of Zoology, Chinese Academy of Sciences (Dr. Heqi Wu, wuhq01@mail.kiz.ac.cn) until genomic DNA extraction. Total genomic DNA was extracted from the cotton swab using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Mitochondrial genomes were sequenced on the GeneMind SURFSeq 5000 platform to generate high-quality, paired-end reads. The mitochondrial genome was assembled using the GetOrganelle v1.7.7.0 program (Jin et al. 2020) with the following parameters: -F animal_mt -R 10. The sequencing depth and coverage map were calculated by means of the method developed by Yang et al. (Ni et al. 2023). The mitogenomes were annotated with MITOS (Bernt et al. 2013) (with manual corrections as needed) and mapped with Organellar Genome DRAW (OGDRAW) 1.3.1 (Greiner et al. 2019). The relative synonymous codon usage (RSCU) and the non-synonymous (Ka) to synonymous (Ks) substitution ratio (Ka/Ks) were computed in PhyloSuite V2 (Zhao et al. 2025). The tRNA secondary structures of transfer RNA (tRNA) were predicted using the tRNAscan-SE program (Chan et al. 2021). Due to the uncertainty surrounding the model selection of the two mitochondrial rRNA genes and the challenges in aligning the short tRNA genes (Gardner et al. 2005; Sullivan and Joyce 2005; Hou et al. 2025), we opted to exclude rRNA- and tRNA-based partitions from our phylogenetic analyses. Phylogenetic inference was performed using the concatenated 13 protein-coding genes (PCGs), aligned by the Multiple Alignment Fasta (MAFFT) program (Katoh and Standley 2013) (v7.505) and trimmed with trimAI program (Capella-Gutierrez et al. 2009). Phylogenetic trees were reconstructed using Maximum Likelihood (ML) and Minimum Evolution (ME) methods. For the ML analysis, IQ-TREE v3.0.1 (Nguyen et al. 2015; Wong et al. 2026) with the built-in ModelFinder (Kalyaanamoorthy et al. 2017) was used to simultaneously determine the optimal partition scheme and substitution models for each partition under the Bayesian Information Criterion (BIC), with automatic merging of partitions sharing similar substitution patterns. The initial partition scheme divided 13 PCGs by codon position, resulting in 39 initial partitions. Maximum likelihood phylogenies were inferred under Edge-unlinked partition model for 5000 ultrafast bootstrap (Minh et al. 2013) replicates, as implemented in PhyloSuite V2 (Zhao et al. 2025). For the ME analysis, MEGA v11.0.13 (Tamura et al. 2021) was used with the Tamura-Nei model with gamma-distributed rate heterogeneity (TN93 + G), and node support was evaluated with 1000 bootstrap replicates. Gallus gallus (NC_007236) was designated as the outgroup for both ML and ME analyses.

Figure 1.

The species reference image is of Hierococcyx sparverioides. The species identification was confirmed in the field by Dr. Heqi Wu, who also took the photograph.

Results

The complete mitochondrial genome of H. sparverioides is a circular molecule of 17,264 base pairs (bp) with an average sequencing depth of 118.64× (Figure 2, Supplementary Figure S1). The gene set under investigation includes the typical set of vertebrate mitochondrial genes. The genome contains a total of 37 genes, of which 28 are encoded on the heavy (H) strand (Figure 2, Supplementary Table S1). Analysis of gene overlap revealed a total of 30 bp across seven junctions (Supplementary Table S1, Supplementary Figure S2). The largest overlap, measuring 10 bp, was identified between ATP8 and ATP6. Several overlaps were found to be as short as 1 bp, including those between trnQ and trnM, trnC and trnY, ATP6 and COX3, and trnS1 and trnL1. The overall nucleotide composition of the sample was found to be 32.9% A, 29.9% C, 13.0% G, and 24.1% T, with a GC content of 42.9%. The AT- and GC-skews of the mitogenome are 0.154 and −0.394, respectively (Supplementary Table S2). These values are consistent with the patterns previously reported for avian mitogenomes (Yu et al. 2021; Hou et al. 2025). The 13 PCGs encompass 11,388 base pairs (approximately 65.96%). The mean AT content across PCGs is 56.51%, ranging from 53.20% in NAD4L to 60.12% in ATP8. The majority of PCGs initiate with the canonical ATG start codon; however, COX I and NAD2 use GTG, and NAD3 uses ATA (Supplementary Table S1). Termination codons include complete TAA, AGG, and AGA, as well as an incomplete T (Supplementary Table S1). RSCU analysis indicates that CUA (Leu1) is the most prevalent codon, while ACG (Thr) is the least used (Supplementary Figure S3, Supplementary Table S3). Calculating the Ka/Ks nucleotide substitution rates revealed that the 13 PCGs in H. sparverioides are primarily characterized by synonymous substitutions (Supplementary Figure S4). The range of Ka/Ks values is from 0.00144146 (COX I) to 0.151491 (NAD6) (Supplementary Table S4). The complete set of 22 tRNA genes exhibits a total length of 1,547 bp and adopts the conventional cloverleaf structure, with the notable exception of trnS1, which is devoid of the DHU arm (Supplementary Figure S5). Phylogenetic trees were reconstructed using both Maximum Likelihood (ML) and Minimum Evolution (ME) methods based on concatenated DNA sequences, yielding largely consistent topologies (Figure 3, Supplementary Figure S6). The newly sequenced PX733929 (H. sparverioides) was placed within the Hierococcyx lineage of the Cuculiformes ingroup (Figure 3). A comparison of previously sequenced complete mitochondrial genomes from other Cuculidae species revealed that H. sparverioides emerged as an independent clade. The phylogenetic analysis indicates that the Hierococcyx lineage and the Cuculus lineage (C. polyiocephalus, C. micropterus, and C. canorus) form a sister relationship, as supported by a bootstrap value of 100 (Figure 3).

Figure 2.

The circular map of the H. sparverioides mitochondrial genome reveals the presence of 13 protein-coding genes, 22 tRNA genes, and two rRNA genes. Functional genes are color-coded and arranged clockwise along the outer circle, and the gray histogram represents GC content with the central line marking the 50% level on the inner circle. Generated using OGDRAW (Greiner et al. 2019).

Figure 3.

The minimum evolution (ME) phylogenetic tree of H. sparverioides is based on mitochondrial nucleotide sequences of 13 PCGs from 9 Cuculiformes taxa and one outgroup. Branch support values are displayed above each branch: ME support values are shown without parentheses; ML support values are shown in parentheses. Branch lengths are indicated below branches. Topologies were generally consistent between methods, except for the position of Urodynamis taitensis and Eudynamys scolopaceus in the ML tree, U. taitensis branches before E. scolopaceus. The following sequences with GeneBank accession numbers were utilized: Hierococcyx sparverioides PX733929 (this study); Centropus bengalensis NC_050049 (Huang et al. 2020); Centropus sinensis KT947122 (Qu et al. 2017); Ceuthmochares aereus NC_052776 (Feng et al. 2020); Cuculus canorus MN067867 (Qiu et al. 2019); Cuculus micropterus MZ048030 (Tian et al. 2021); Cuculus poliocephalus NC_028414 (Wang et al. 2016); Eudynamys scolopaceus NC_060520 (Lee et al. 2022); Urodynamis taitensis NC_011709 (Pratt et al. 2009) and Gallus gallus NC_007236 (Nishibori et al. 2005).

Discussion and conclusion

The mitochondrial genome of H. sparverioides is a circular double-stranded DNA molecule that is 17,264 base pairs (bp) in length. This genome contains 13 PCGs, 22 tRNAs, 2 rRNAs, and a control region. This observation aligns with the majority of known vertebrates, as evidenced by studies (Boore 1999; Yang et al. 2010; Man et al. 2011; Wei et al. 2011). The structural characteristics of H. sparverioides align with typical patterns observed in the Cuculidae and other avian lineages (Wang et al. 2022; Hou et al. 2025). In the ancestral avian arrangement, the control region (CR) is positioned between trnE and trnF (Mindell et al. 1998). In contrast, H. sparverioides possesses a functional CR (1,475 bp) located between trnT and trnP, and a presumably noncoding (nc) sequence (222 bp) between trnE and trnF, a derived arrangement relative to the ancestral avian gene order characterized in Gallus gallus (Desjardins and Morais 1990). This arrangement is shared with other Cuculidae species, including C. canorus (MN067867), which similarly contains a functional CR (1,265 bp) between trnT and trnP and a short nc region (306 bp) between trnE and trnF (Qiu et al. 2019). The presence of this derived arrangement in Cuculidae suggests that the CR duplication event predates the divergence of this family, with subsequent degeneration of CR₂ producing the remnant pattern observed in extant cuckoo species. Comparison of the CR compositional biases of H. sparverioides (AT-skew = −0.001; GC-skew = −0.382) with warbler species (Singh et al. 2008) bearing duplicated CR gene orders (AT-skew ranging from −0.024 to 0.003; GC-skew ranging from −0.309 to −0.392) and the single CR of the remnant CR₂ species C. crassirostris (AT-skew: −0.108, GC-skew: −0.355) reveals no distinct skew pattern distinguishing CR evolutionary states, suggesting that H-strand compositional bias is an intrinsic property of the CR maintained regardless of duplication or degeneration status.

As is well-documented in the relevant literature, AT-skew, GC-skew, and A + T content are commonly used to assess differences in the composition of nucleotides in mitochondrial genomes (Hassanin et al. 2005; Zhang et al. 2025). This mitogenome exhibits a higher AT content (57.1%) compared to its GC content (42.9%), a feature that is analogous to that observed in other species of Cuculidae (Wang et al. 2022; Zhang et al. 2025). Consistent with the well-established pattern that functional mitochondrial genes generally evolve under purifying selection, the Ka/Ks ratios of all 13 PCGs were significantly less than 1, confirming that these genes are subject to strong purifying selection and maintain conserved functions typical of avian mitogenomes (Hurst 2002; Hou et al. 2025). Phylogenetic analyses clearly demonstrate that H. sparverioides is most closely related to the Cuculus within Cuculidae, in accordance with morphology-based classifications. In both Minimum Evolution (ME) and Maximum Likelihood (ML) trees, H. sparverioides was recovered as the sister taxon to the Cuculus with robust support, forming an independent lineage distinct from Cuculus. Despite their rapid evolutionary rate, mitogenomes retain species-specific phylogenetic signals that enable reliable resolution of such relationships. The mitogenome of H. sparverioides presented here thus contributes to both the systematics and molecular identification of Cuculidae.

This study presents the first report on the complete mitochondrial genome sequence of H. sparverioides, accompanied by a comprehensive structural analysis. The mitogenomic data provide molecular support for the phylogenetic placement of H. sparverioides within Cuculiformes. Brood parasitism is a striking life-history strategy that has also been characterized mitogenomically in other parasitic cuckoos, such as C. canorus (Qiu et al. 2019), E.scolopaceus (Lee et al. 2022), and C. coromandus (Zhanget al.2025), as well as in several non-cuculid avian lineages.Because mitochondria are central to energy metabolism, the distinctive reproductive and behavioural traits associated with obligate brood parasitism may impose lineage-specific selective pressures on mitochondrial genes, although how such pressures translate into mitogenomic variation remains largely unexplored. Comparing Ka/Ks ratios between parasitic and non-parasitic lineages across birds therefore offers a promising avenue to test whether obligate parasitism imposes distinct selective pressures on mitochondrial genes, and expanding taxon sampling will help determine whether brood parasitism originated once or multiple times.

Funding Statement

This work was supported by the National Key R&D Program of China, Grant No. 2022YFC2602500 and the Chinese Bird Diversity Observation Network.

Ethical approval

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Kunming Institute of Zoology, Chinese Academy of Sciences (Approval：IACUC-OE-2021-08-004). At the time of sample collection, all procedures were conducted in compliance with relevant guidelines and the ARRIVE guidelines.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The genome sequence data supporting the findings of this study are openly available in GenBank (NCBI) at https://www.ncbi.nlm.nih.gov under accession PX733929. The associated BioProject, BioSample, and SRA numbers are PRJNA1521087, SAMN62788327, and SRR40418034, respectively.