Hystricidae (the Old World porcupines), which includes three distinguishable genera (Hystrix, Atherurus, Trichys), is one of the four families contained in the infraorder Phiomorpha. Phylogenetic evidence led to the hypothesis that the genera Trichys and Atherurus originated in the Palaeocene from an unknown Hystricognath ancestor in Asia. However, the earliest Porcupine fossils were recorded only in the Miocene in Europe. The extant Hystricidae species are distributed in Asia, Africa and Europe (only in Italy). Based on their habitat environment, Porcupines are generally adapted to tropical and subtropical climate.
In China, two species from two genera exist, i.e. Atherurus macrourus (Brush-tailed Porcupine) and Hystrix brachyura (Malayan Porcupine). The latitude range of Hystrix brachyura is 5°S–35°N, which totally encompasses the range of Atherurus macrourus, i.e. 1°N–31°N. A large number of Hystrix fossils have been excavated from Chinese Quaternary strata. The oldest Porcupine discovered in Asia, Hystrix lufengensis, has been dated to the Late Miocene (8 million years ago) and was located in Yunnan Province, Southern China. Fossils are particularly abundant in southern China compared to northern China, with fossil findings reported in nearly all provinces in subtropical areas from the Late Miocene to the Holocene. Nevertheless, typical Porcupine fossils are present in northern China throughout the whole Quaternary. Among all northern excavation sites, Zhoukoudian, Beijing has the richest assemblages of northern Porcupine fossils and represents the latest known record before Hystrix finally disappeared from the Beijing area.
Morphologically, Quaternary Porcupine fossils are quite distinct from extant species. Clarifying the evolutionary history of Porcupines is of great significance to better understand their response to climate change during their geographic extirpation in northern China during the Quaternary. However, molecular phylogenetic relationships between fossils and extant Porcupines remain unclear.
In a paper published in the journal BMC Evolutionary Biology on 18 July 2020, Guilian Sheng of the School of Environmental Studies and State Key Laboratory of Biogeology and Environmental Geology at the China University of Geosciences, Jiaming Hu, also of the School of Environmental Studies China University of Geosciences, Haowen Tong of the Key Laboratory of Vertebrate Evolution and Human Origins at the Institute of Vertebrate Paleontology and Paleoanthropology of the Chinese Academy of Sciences, Bastien Llamas of the Australian Centre for Ancient DNA at the University of Adelaide, Junxia Yuan of the Faculty of Materials Science and Chemistry at the China University of Geosciences, Xindong Hou, again of the School of Environmental Studies China University of Geosciences, Shungang Chen, also of the Faculty of Materials Science and Chemistry at the China University of Geosciences, Bo Xiao, once again of the School of Environmental Studies China University of Geosciences, and Xulong Lai, also of the State Key Laboratory of Biogeology and the Faculty of Earth Sciences at the China University of Geosciences, present the results of a study in which they extracted ancient DNA and sequenced partial cytochrome b (cyt b) and 12S ribosomal ribonucletic acid (rRNA) genes from Late Pleistocene Hystricidae fossils/sub-fossils in China.
Sheng et al. constructed a phylogenetic tree to identify the evolutionary status of these fossils/sub-fossils and further discussed mechanisms underlying the range contraction of Porcupines.
Via multiple DNA (deoxyribonucleic acid) extractions, multiple amplifications, cloning of amplification products, sequencing of multiple clones, and replication of results by an independent laboratory, Sheng et al. analysed seven Late Pleistocene fossil and sub-fossil Porcupines: two from Tianyuan Cave, Zhoukoudian, Beijing (Specimens Nos.: CADG-67, CADG-68; 30 500–4670 years old); two from Fuyan Cave, Daoxian County in Hunan province (Specimens Nos.: CADG-69, CADG-70; 153 800–129 600 years old), and three from Loushan Cave, Wuming County in Guangxi province (Specimens Nos.: CADG-71, CADG-72, CADG-73; 24 000-13 000 years old), respectively. Seven overlapping fragments of the mitochondrial cyt b gene ranging from 90 to 111 base pairs (excluding primers) were amplified and sequenced for the two specimens from Tianyuan Cave at Zhoukoudian in Beijing (525 base pairs total for CADG-67, GenBank Accession No: MK579190; 605 bbase pairs p for CADG-68, GenBank Accession No: MK579191). Another five overlapping fragments of the 12S rRNA gene ranging from 66 to 186 base pairs (excluding primers) were amplified and sequenced for the same samples (601 base pairs for CADG-67, GenBank Accession No: MK419154; 573 base pairs for CADG-68, GenBank Accession No: MK419155). Identical sequences were obtained when experiments were independently replicated in the State Key Laboratory for Biogeology and Environmental Geology at the China University of Geosciences (Wuhan) and Australian Centre for Ancient DNA at The University of Adelaide. In contrast, no fragments could be recovered from five samples collected from Hunan Province and Guangxi Province despite repeated attempts. No sequences were successfully amplified from all extraction and polymerase chain reaction blank controls. Eventually, Sheng et al. considered only Tianyuan Cave samples for further phylogenetic analyses.
To investigate the phylogenetic position of the fossil Porcupines in the Hystricidae family, we downloaded various sequences from GenBank and composed three datasets to use the available sequences as much as it is possible.
The relationship between Tianyuan Cave Porcupines, Hystrix and Atherurus was inferred using Dataset 1 (446 base pairs of cyt b gene) in a Maximum Likelihood tree. Tianyuan Cave Porcupines form a monophyletic clade basal to all Hystrix cristata and Hystrix africaeaustralis. Atherurus macrourus is sister to all Hystrix and Tianyuan Cave samples. Branch lengths suggest that the Late Pleistocene Chinese Porcupines are more related to Hystrix than to Atherurus.
The molecular phylogeny of all species in the Hystricidae family with molecular data available was reconstructed using Dataset 2 (204 base pairs of cyt b gene) in a Maximum Likelihood analysis. Individuals from three genera, i.e. Atherurus, Hystrix, and Trichys, form distinct groups, with Trichys standing basal in the tree. The two Late Pleistocene Chinese Porcupines fall within the Hystrix group and are closely related to one Hystrix brachyura individual from Thailand (GenBank Accession No: JQ991599). Although the relationship between Hystrix brachyura from Peninsular Malaysia or Borneo and Hystrix indica from India is generally poorly resolved, Sheng et al. confidently reject the hypothesis that the Tianyuan Cave Porcupines are either Hystrix cristata or Hystrix africaeaustralis.
Results using Dataset 3 in a Maximum Likelihood analysis based upon molecular phylogeny based on 12S rRNA are consistent with the phylogeny obtained from the partial cyt b gene data set, except for one Hystrix indica individual (GenBank Accession No: FJ472546) that falls basal in the family Hystricidae. The Tianyuan Cave Porcupines form a monophyletic group and have a well-supported sister relationship with a Hystrix brachyura individual (bootstrap value of 70%).
Hydrolysis, oxidation and microbial action are the main factors that directly cause DNA fragmentation and degradation. Therefore, the preservation of ancient DNA molecules is not obviously related to age of samples, whereas temperature, pH, ionic strength and humic acid content of the burial environment directly affect the degradation rate of ancient DNA. Chronologically, all Porcupine samples in Sheng et al.'s study have similar ages and have been dated to the Late Quaternary. However, they only successfully amplified ancient DNA fragments from specimens collected from Tianyuan Cave, while no positive results were obtained from the other two caves despite multiple attempts. Sediments in Tianyuan Cave have been deposited since 42 000 years ago and experienced four stages that correspond to four different climatic conditions. The layer of Sheng et al.'s Porcupine samples was at the third Stage, a cold and dry stage from the late Pleistocene to the early Holocene. Although they have no accurate information to estimate the palaeo-environment of the sampling sites in Hunan and Guangxi provinces, it is clear that samples in these two sites were discovered in wet soil. Sheng et al. assume that the warm and humid environments in Hunan and Guangxi provinces accelerated DNA decay to the point of DNA fragments being too short for efficient polymerase chain reaction amplification using our primer sets. Alternatively, it is possible that polymerase chain reaction inhibitors present in the soil were co-extracted with DNA, resulting in failure to amplify DNA. Of note, partial and even complete ancient mitochondrial DNA sequences have been successfully retrieved from megafauna samples collected from southern China. It is possible that the small Porcupine bones did not preserve DNA as efficiently as in large Mammal bones. In contrast, Tianyuan Cave is located in northern China, where the general cold and dry climate conditions are likely to better preserve ancient DNA for polymerase chain reaction-based analyses.
Previous morphological identification named Tianyuan Cave Porcupines as Hystrix subcristata. There are various opinions with regard to the classification of Hystrix in China. A 1995 study sorted Chinese Hystrix into three species: Hystrix indica, Hystrix hodgsoni, and Hystrix brachyura. Dees van Weers further classified Hystrix brachyura into two subspecies: Hystrix brachyura subcristata and Hystrix brachyura yunnanensis. Instead of the subspecies status, Haowen Tong restored the original classification status of Hystrix subcristata into a species level, since differences in measurements of skull height and nasal length/occipito-nasal length of Hystrix subcristata and Hystrix brachyura are indeed obvious. In Sheng et al.'s study, both cyt b and 12S rRNA gene analysis support that Tianyuan Cave Porcupines have a closer relationship with Hystrix than with other genera in the family Hystricidae. However, some poorly resolved nodes within the Hystrix clade lead to an ambiguous phylogenetic assignment of the Tianyuan Cave Porcupines at the species level. Sheng et al. say with confidence that the Tianyuan Cave Porcupines are not Hystrix cristata or Hystrix africaeaustralis, and that they are likely more closely related to Hystrix brachyura. Homologous sequences from morphologically distinct Hystrix brachyura subcristata, Hystrix brachyura yunnanensis, or Hystrix subcristata individuals are not available yet. Therefore, it seems that the taxonomic assignment of the Late Pleistocene Porcupines in Tianyuan Cave as a subspecies or a species remains an open question in terms of molecular evidence. Further palaeogenomic evidences from more contemporary and fossil individuals may help to solve this taxonomic issue.
Among the family Hystricidae, the oldest genera Trichys have the smallest body size and the most rat-like appearance. This genus is distributed in tropical areas near the equator such as Indonesia and the Malay Peninsula. The evolutionarily younger genera Atherurus and Hystrix are distributed in higher northern latitudes such as in Central Asia and Southern Europe. Therefore, the contemporary Hystricidae members include species adapted to warm climate in both tropical and subtropical zones. The Tianyuan Cave Porcupines were located at 39° N in terms of latitude, while the distribution range of wild Hystrix brachyura is generally no more than 35° N. Moreover, the Tianyuan Cave samples are more closely related to one individual from Thailand (GenBank Accession No: JQ991599) than to individuals from Malaysia (GenBank Accession Nos: KX580791–5). The associated fauna discovered together with Tianyuan Cave Porcupine samples, such as Macaca mulatta, Ursus thibetanus, Arctonyx collaris, Paguma larvata, Panthera pardus, etc., are all warmth-adapted species. These warmth-adapted species, together with the phylogenetic relationship revealed by this study, indicate that mid-Holocene extirpation events have occurred in the evolutionary history of Porcupines.
Many palaeontologists indicated that the skeletal characteristics of Porcupine are related to their evolutionary status and habitat environment. Wang Ban-Yue and Qiu Zhan-Xiang compared eight species in the genera Hystrix that are distributed from Indonesia to central China and suggested that Hystrix seems to have the following evolutionary tendencies: the rostrum heightens and the nasal and frontal bones enlarge as latitude increases. By comparing the nasal length and other morphological traits between Hystrix species, Haowen Tong discovered that the skull of Hystrix in the tropics is rounder, while those in northern areas have longer nasal length. For instance, the two extant Hystrix (Hystrix cristata and Hystrix brachyura subcristata), which have the longest nasal length, are distributed in relatively higher latitudes. It seems that Tianyuan Cave porcupines did have morphological characteristics making them to be better adapted to live in northern China. Cold climate indicator such as the loess record suggests that frequent paleoclimate fluctuations occurred during the Late Pleistocene in China. This can also be verified by the coincidence of the frequent and dramatic sea level rises and falls during the same period.
Therefore, Sheng et al. hypothesize that porcupines have migrated to northern China during a warm period between the Late Miocene and the Early Pleistocene, and morphologically adapted to the relatively colder climate during the Late Pleistocene. They may have become locally extinct in the north during the Mid Holocene while the southern populations survived and formed the contemporary population distribution.
In Sheng et al.'s study, they successfully analysed partial cyt b and 12S rRNA gene sequences from Tianyuan Cave Porcupines. Phylogenetic analyses support a possible close relationship between our Late Pleistocene samples and extant Hystrix brachyura, which are distributed in tropical and subtropical areas of Southeast and South Asia. Together with morphological adaptation characteristics and climate change evidence, our molecular results reveal a northern extirpation of porcupines during the Holocene.
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