Парагогенгейм. Лето Парагогенгейма

Индийские наги, съедающие мунтжака.

```
```

Диплоидный Кариотип - ранее разросшейся (вокруг ещё более древлее поселённой сосны) берёзы из 84 хромосом вновь принимает в свой биоценоз кариотип 24 хромосом ели и сосны, раздробившейся своим кариотипом в берёзе:

Корни в небо полетели -
Маки маковкой запели!.. -:

Сравнение ценофлор сосновых и берёзовых лесов Гунибского плато (Дагестан)
Кессель Д.С.1
, Абдурахманова З.И.2
, Щукина К.В.1
, Гаджиатаев М.Г.2
1Ботанический институт им. В.Л. Комарова РАН, Санкт-Петербург, dasha_kessel@mail.ru
vyatka_ks_72@mail.ru 2Горный ботанический сад – ОП Дагестанского федерального
исследовательского центра РАН, г. Махачкала, zagidat.abdurahmanova88@mail.ru
gadzhiataev@mail.ru
Гунибское плато расположено в северо-западной части известнякового Внутригорного
Дагестана на высотах от 1400 до 2354 м над ур. м., общей площадью около 36 км2
. Почвы
представлены каменисто-щебнистыми, маломощными коричневыми лесными и горнолуговыми черноземовидными; преобладают карбонатные почвообразующие породы.
Сосновые (Pinus kochiana Klotsch ex G. Koch.) и берёзовые (Betula litwinowii Doluch. и В.
raddeana Trautv) леса на Гунибском плато встречаются от нижней границы лесного пояса до
верхних пределов распространения леса (1400-2100 м над ур. м.). Сосняки и березняки
приурочены к склонам северной экспозиции, образуют чистые массивы в верхней части плато
VIII Всероссийская конференция с международным участием «Горные экосистемы и их компоненты», Нальчик – 2021
37
(на высотах до 2100 м над ур. м.). Кроме того, сосна Коха входит в состав берёзовых лесов
плато. Небольшие участки молодняков сосны формируются и на южном склоне.
Во флоре сосудистых растений Гунибского плато насчитывается 657 видов (Омарова,
2013). В сообществах сосняков Гунибского плато выявлено 226 видов, относящихся к 147
родам и 49 семействам (Абдурахманова, Садыкова, 2015). Ценофлора березняков включает
251 вид, 164 рода, 63 семейства. Общими для сообществ сосновых и берёзовых лесов являются
только 130 видов.
Спектр ведущих семейств Гунибского плато относится к Fabaceae-типу, характерному
для Средиземноморской группы. Ценофлоры сосновых и берёзовых лесов представлены
Rosaceae-типом, характеризующим среднеевропейскую группу. На долю преобладающих по
количеству видов 6 семейств приходится 50% видов ценофлор сосняков и березняков (113 и
129 видов соответственно) (Хохряков, 2000).
По количеству видов в сосновых лесах лидирует сем. Asteraceae, включающее 32 вида;
на втором месте находится сем. Poaceae (25 видов). Семейство Rosaceae с 18 видами занимает
третье место в спектре ведущих семейств, a Fabaceae с 14 видами занимает 4 место; за ним
следуют Apiaceae (13 видов) и Lamiaceae (13 видов) (Абдурахманова, Садыкова, 2015). Для
березняков этот ряд выглядит следующим образом: сем. Asteraceae – 33 вида, Rosaceae – 26
видов, Poaceae – 25 видов, Apiaceae – 16 видов, Fabaceae – 15 видов, Lamiaceae – 13 видов.
При сравнении ценофлор сосновых и берёзовых лесов плато выявлено, что в березняках
произрастает большее количество видов, относящихся к лесному ценотипу: 29% лесных видов
от общего количества видов в березняках и 21% – в сосняках. Видов, характерных для лугов,
в том числе субальпийских и альпийских, в сосновых лесах больше, чем в берёзовых (64% и
57% соответственно). Также в сосняках выявлено большее количество рудеральных видов
(4,5%), чем в березняках (2,5%).
На основании проведённого анализа можно заключить, что, хотя берёзовые и сосновые
леса на Гунибском плато произрастают в сходных местообитаниях, их ниши несколько
расходятся при крайних значениях свето- и влагообеспеченности. Березняки более устойчивы
к затенению и могут произрастать на крутых северных склонах с максимальным затенением.
А сосняки более устойчивы к засухе и могут занимать участки даже на южных склонах.

VIII Всероссийская конференция с международным участием «Горные экосистемы и их компоненты», Нальчик – 2021
20
Микробиологическая активность бурых лесных почв северного макросклона
Западного Кавказа
Улигова Т.С., Горобцова О.Н., Гедгафова Ф.В., Темботов Р.Х., Хакунова Е.М.,
Баккуева З.Л.
Институт экологии горных территорий имени А.К. Темботова РАН, г. Нальчик,
ecology_lab@mail.ru
На территории двух памятников природы северного макросклона Западного Кавказа –
«Гуамское ущелье» и «Массив самшита колхидского» в бассейне р. Цице (Майкопский район
Республики Адыгея) сохранились природные лесные биогеоценозы, изучение которых
позволяет определить эталонные показатели, необходимые для мониторинга горных
экосистем в условиях возрастающей антропогенной нагрузки и климатических изменений. В
пределах высот 447-897 м над ур. м. изучали следующие типы колхидских лесов: 1. грабоворазнотравный; 2. ясенево-пихтово-буковый; 3. грушево-буковый; 4. грабово-буковый; 5.
грабово-буковый.

~~~
~~~

И деревья полетели -
Сосны с берёзами запели:

Хорошо сосне с берёзой:
Ей расти так проще с Розой!..


В Дагестане много бука -
Не встречается разлука.

Лука - слёз там не встречаешь -
Лук Лукавый - вылукаешь!!!


Лето Леса Лепота -
Чело Века Чистота.

Зело Снега Хрустота -
Жертв Снеданья - Пустота.

~~~
~~~

Четвероногие - тетраподы, - в том числе включающие птиц, - надкласса челюстноротых, из группы костных позвоночных, якобы произошедших от рыб, "входящих" же (?!)в этот же класс "челюстноротых":

Четвероногие летают, -
А по мне так - птицы ходят и сползают.

Ведь, и комары из вод не подлетают
Кровь испить не подползают?

Рассмотрите птицу Додо, с плодоносяще-тростниково-сахародающего острова Маврикий, лишённую летающих крыльев. К этой птице добрые люди подходили и били палкой по голове, благодарили её и пожирали же её самую.

Не могу исключить эволюции летательного аппарата крыльев из задней пары конечностей, представляя полёт такого существа не иначе, чем пузом кверху и клоакой вперёд, при том, что для женщин - это существо-производитель мужской линии гамет, а для мужчин - наоборот, святая самка Богомола.

Здесь же добавим, при том что по количеству - диплоидному набору (кариотипа) - хромосом птицы (куры - 78, голуби до 80) имеют близко к большому (большему, чем у человека 46) числу хромосом, как и быки (60).

Отметим (Актуальные вопросы цитогеномики, организации и эволюции геномов и хромосом у птиц

Романов М.Н.,1,2 Трухина А.В.,3 Смирнов А.Ф.,3 Гриффин Д.К.2

1ФГБОУ ВО «Московская государственная академия ветеринарной медицины и биотехнологии – МВА имени К. И. Скрябина», Москва, Россия;

2Университет Кента, Кентербери, Великобритания;

3ФГБОУ ВО «Санкт-Петербургский государственный университет», Санкт-Петербург, Россия
E-mail: m.romanov@kent.ac.uk):

Перечень секвенированных геномов птиц в базе данных NCBI (по состоянию 09.08.2021 г.)

№ п/п Вид Латинское название Отряд Число хромосом в геномной сборке Идентификатор генома в базе данных NCBI Геном митохондрии
1 Американский аист Ciconia maguari Аистообразные 31 92799 секвенирован
2 Калифорнийский кондор Gymnogyps californianus Американские грифы 30 730 –
3 Американская пищуха Certhia americana Воробьинообразные 24 103027 –
4 Белая трясогузка Motacilla alba Воробьинообразные 31 43097 секвенирован
5 Белогрудая гологлазая муравьянка Rhegmatorhina hoffmannsi Воробьинообразные 35 92741 –
6 Большая синица Parus major Воробьинообразные 31 12863 секвенирован
7 Буроголовый коровий трупиал Molothrus ater Воробьинообразные 34 88920 –
8 Галка Coloeus monedula Воробьинообразные 29 93095 –
9 Деревенская ласточка Hirundo rustica Воробьинообразные 39 73420 –
10 Домовый воробей Passer domesticus Воробьинообразные 30 17653 секвенирован
11 Дрозд Свенсона Catharus ustulatus Воробьинообразные 43 86530 секвенирован
12 Желтогорлый певун Geothlypis trichas Воробьинообразные 34 86337 –
13 Зарянка Erithacus rubecula Воробьинообразные 33 92589 –
14 Зебровая амадина Taeniopygia guttata Воробьинообразные 41 367 секвенирован
15 Зяблик Fringilla coelebs Воробьинообразные 30 34546 –
16 Малый древесный вьюрок Camarhynchus parvulus Воробьинообразные 31 84210 –
17 Миртовый лесной певун Setophaga coronata Воробьинообразные 31 46404 секвенирован
18 Мухоловка-белошейка Ficedula albicollis Воробьинообразные 30 11872 секвенирован
19 Новокаледонский ворон Corvus moneduloides Воробьинообразные 36 85337 секвенирован
20 Острохвостая бронзовая амадина Lonchura striata Воробьинообразные 31 43765 секвенирован
21 Острохвостый красноногий манакин Chiroxiphia lanceolata Воробьинообразные 35 86579 –
22 Прекрасный расписной малюр Malurus cyaneus Воробьинообразные 25 86232 секвенирован
23 Садовая славка Sylvia borin Воробьинообразные 37 92826 –
24 Серая ворона Corvus cornix Воробьинообразные 29 18230 секвенирован
25 Стрелок Acanthisitta chloris Воробьинообразные 38 32002 секвенирован
26 Черноголовая славка Sylvia atricapilla Воробьинообразные 35 8421 –
27 Черногорлый цветокол Diglossa brunneiventris Воробьинообразные 31 103900 –
28 Черношапочная гаичка Poecile atricapillus Воробьинообразные 19 33953 секвенирован
29 Обыкновенная горлица Streptopelia turtur Голубеобразные 33 81804 –
30 Сизый голубь Columba livia Голубеобразные 29 10719 секвенирован
31 Американская савка O***ra jamaicensis Гусеобразные 34 87936 секвенирован
32 Африканский блестящий чирок Nettapus auritus Гусеобразные 34 87938 секвенирован
33 Крапчатая утка Stictonetta naevosa Гусеобразные 34 87939 секвенирован
34 Кряква (домашняя утка) Anas platyrhynchos Гусеобразные 41 2793 секвенирован
35 Лебедь-шипун Cygnus olor Гусеобразные 36 38225 секвенирован
36 Мускусная утка Cairina moschata Гусеобразные 30 8552 –
37 Хохлатая чернеть Aythya fuligula Гусеобразные 36 33654 секвенирован
38 Черноголовая утка Heteronetta atricapilla Гусеобразные 34 87937 секвенирован
39 Золотой шилоклювый дятел Colaptes auratus Дятлообразные 12 96575 –
40 Краснолобый медник Pogoniulus pusillus Дятлообразные 46 96564 секвенирован
41 Пушистый дятел Picoides pubescens Дятлообразные 46 32059 секвенирован
42 Восточный венценосный журавль Balearica regulorum Журавлеобразные 37 17144 секвенирован
43 Эму Dromaius novaehollandiae Казуарообразные 31 123 секвенирован
44 Хохлатая кариама Cariama cristata Кариамообразные 52 31967 секвенирован
45 Исполинский козодой Nyctibius grandis Козодоеобразные 38 92333 секвенирован
46 Обыкновенный козодой Caprimulgus europaeus Козодоеобразные 37 101473 –
47 Обыкновенная кукушка Cuculus canorus Кукушкообразные 41 32170 секвенирован
48 Банкивская джунглевая курица (домашняя курица) Gallus gallus Курообразные 41 111 секвенирован
49 Индейка Meleagris gallopavo Курообразные 36 112 секвенирован
50 Немой перепел (японский перепел) Coturnix japonica Курообразные 29 113 секвенирован
51 Обыкновенная цесарка Numida meleagris Курообразные 30 14094 секвенирован
52 Волнистый попугайчик Melopsittacus undulatus Попугаеобразные 32 10765 секвенирован
53 Какапо Strigops habroptila Попугаеобразные 25 75115 секвенирован
54 Калита Myiopsitta monachus Попугаеобразные 25 40151 –
55 Синелобый амазон Amazona aestiva Попугаеобразные 29 40915 –
56 Абиссинский рогатый ворон Bucorvus abyssinicus Птицы-носороги 41 86364 –
57 Нубийская щурка Merops nubicus Ракшеобразные 36 31978 секвенирован
58 Гагарка Alca torda Ржанкообразные 26 84534 секвенирован
59 Золотистая ржанка Pluvialis apricaria Ржанкообразные 38 100067 секвенирован
60 Речная крачка Sterna hirundo Ржанкообразные 27 66333 секвенирован
61 Желтогорлый рябок Pterocles gutturalis Рябкообразные 36 32063 секвенирован
62 Кречет Falco rusticolus Соколообразные 24 43830 секвенирован
63 Сапсан Falco peregrinus Соколообразные 19 132 секвенирован
64 Степная пустельга Falco naumanni Соколообразные 27 44448 секвенирован
65 Калипта Анны Calypte anna Стрижеобразные 33 32060 секвенирован
66 Краснохохлый турако Tauraco erythrolophus Туракообразные 33 32247 –
67 Красный фламинго Phoenicopterus ruber Фламингообразные 33 31928 –
68 Беркут Aquila chrysaetos Ястребообразные 28 32031 –

Из чего следует, что фермионно-подобное (по нечётному количеству равенства числа хромосом - нечётному количеству протонов в фермионах) число хромосом некоторых птиц и богомола-самки (27) связано с определёнными поведенческими особянностями того или иного вида. Здесь возможно указать только либо на агрессивно-ауто-интро-видовые особенности (самка-богомол) либо, даже на травоядно-прогрессивные особенности видовых разнообразий с пониженным числом хромосом в кариотипе у Индийского мунтжака с 7 хромосомами у мужских особей и 6 хромосомами у - женских.
~~~~~~
Вообще же, увеличение количества хромосом создаёт относительно антропо-фитные особенности поведения животного:
$
[60 хромосом у коровы и зубра, 78 хромосом - у собаки:]$
~~~~~~

An extraordinarily stable karyotype of the woody Populus species revealed by chromosome painting
Haoyang Xin,Tao Zhang,Yufeng Wu,Wenli Zhang,Pingdong Zhang,Mengli Xi,Jiming Jiang
First published: 17 September 2019

~~~~~~
doi.org/10.1111/tpj.14536

~~~~~~
Citations: 26
SECTIONSPDFPDFTOOLS SHARE
Summary
The karyotype represents the basic genetic make-up of a eukaryotic species. Comparative cytogenetic analysis of related species based on individually identified chromosomes has been conducted in only a few plant groups and not yet in woody plants. We have developed a complete set of 19 chromosome painting probes based on the reference genome of the model woody plant Populus trichocarpa. Using sequential fluorescence in situ hybridization we were able to identify all poplar chromosomes in the same metaphase cells, which led to the development of poplar karyotypes based on individually identified chromosomes. We demonstrate that five Populus species, belonging to five different sections within Populus, have maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements were observed on any of the 19 chromosomes among the five species. Thus, the chromosomal synteny in Populus has been remarkably maintained after nearly 14 million years of divergence. We propose that the karyotypes of woody species are more stable than those of herbaceous plants since it may take a longer period of time for woody plants to fix chromosome number or structural variants in natural populations.

Introduction
The number and appearance of all chromosomes in a eukaryotic species is described as the karyotype; this provides the most basic genomic information and can be used to display the phylogenetic relationships and evolutionary origins among related species. Genetically related species often share a similar karyotype. However, karyotype evolution is associated with distinct patterns in different evolutionary lineages. For example, Solanaceae, a plant family containing several major food crops including potato, tomato and eggplant, diverged about 40 million years (Myr) ago from an ancestral diploid species with 2n = 24 chromosomes. Nearly all diploid family members have maintained this chromosome number (Wu et al., 2006). Potato, tomato and eggplant, which diverged about 15 Myr ago (Wu and Tanksley, 2010), have maintained a similar karyotype (Braz et al., 2018). In contrast, plant species in the family Brassicaceae are known to have highly variable basic chromosome numbers and have undergone massive chromosomal rearrangements (Mandakova et al., 2017). Accurate karyotyping as well as genome sequencing have been conducted in only a limited number of plant species. The molecular mechanisms that govern karyotype evolution have remained elusive.

The identification of individual chromosomes is the foundation for developing a karyotype. Various cytogenetic techniques have been developed for identifying chromosomes, and fluorescence in situ hybridization (FISH) has become the most important technique for identifying chromosomes in plants (Jiang et al., 1995; Cheng et al., 2001; Lysak et al., 2001; Kato et al., 2004; Jiang and Gill, 2006). However, FISH-based chromosome identification systems have not been established in most non-model plant species due to a lack of robust DNA probes (Jiang, 2019). The recent development and application of synthetic oligonucleotide (oligo) probes has made it possible to identify chromosomes in plant species with only limited genomic resources (Jiang, 2019). Oligos specific to an entire chromosome can be identified computationally, synthesized in parallel as a pool and labeled as a ‘chromosome painting’ probe (Beliveau et al., 2012; Han et al., 2015). Chromosome painting allows the identification and tracking of the target chromosome at different stages of mitosis and meiosis (Han et al., 2015). Oligo-based chromosome painting probes have been developed in an increasing number of plant species (Han et al., 2015; Qu et al., 2017; Braz et al., 2018; He et al., 2018; Hou et al., 2018; Meng et al., 2018; Albert et al., 2019), and have been used in various cytogenetic and genome studies (Jiang, 2019).

The genus Populus is composed of dioecious woody plants which belong to the family Salicaceae. Cytological studies showed that Populus predominantly comprises diploids with a basic haploid chromosome number of 19 (Blackburn and Harrison, 1924). Most chromosomes of Populus species are small and of a similar size, making it impossible to identify chromosomes individually (Ribeiro et al., 2008; Islam-Faridi et al., 2009). A karyotype has been reported for a few Populus species (Zhang et al., 2005; Dong et al., 2007; Hu et al., 2012); however, chromosomes were not individually identified in these previous reports. Thus, these karyotypes developed from different Populus species cannot be compared with each other. We have previously developed an oligo-based painting probe for chromosome 19 in Populus trichocarpa (Xin et al., 2018). Here we report a complete set of painting probes for all 19 chromosomes of P. trichocarpa. Using these painting probes and a sequential FISH procedure we were able to identify all 19 chromosomes in the same metaphase cells in poplar species. This technique allowed development of accurate karyotypes based on individually identified chromosomes. We demonstrate that five different Populus species, belonging to five different sections of Populus, show a super-conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the 19 chromosomes among the five species. Thus, the chromosomal level of genetic synteny has been maintained after about 14 Myr of divergence of these woody species. We also demonstrate various applications of the chromosome painting probes in genetics and genomic studies in poplar.

Results
Development of chromosome-specific painting probes in poplar
To develop chromosome painting probes in poplar we computationally identified all single-copy oligos [45 nucleotides (nt)] from each of the 19 pseudomolecules of the P. trichocarpa genome (version 3.0, http://www.phytozome.net/poplar) using Chorus2 (https://github.com/zhangtaolab/Chorus2) (see Experimental Procedures). We selected approximately 25 000 oligos associated with each poplar chromosome, ranging from 24 448 for chromosome 16 to 26 929 for chromosome 19 (Data S1 in the online Supporting Information). The 19 oligo libraries were synthesized individually to develop 19 chromosome painting probes. Oligos for each chromosome were selected to uniformly cover the entire length of the chromosomes, except for the genomic regions containing highly repetitive DNA sequences, including the centromeres (Figure S1), to ensure that painting probes generated uniform signals on the chromosomes.

Chromosome painting in different poplar species
We first examined the quality of the chromosome painting probes in P. trichocarpa (section Tacamahaca). Pairs of painting probes were labeled with biotin and digoxigenin, respectively (Han et al., 2015), and hybridized to the somatic metaphase chromosomes prepared from P. trichocarpa. Each of the 19 probes generated bright and chromosome-specific signals on P. trichocarpa chromosomes (Figure 1). However, the distal ends on the short arms of chromosomes 8 and 14 were not covered by the painting probes (Figure 1f,j), although the oligos were selected throughout the two pseudomolecules (Figure S1). Similarly, we previously reported that the distal end of the short arm of chromosome 19, the sex chromosome in poplar, was not covered by the reference genome based on painting analysis of chromosome 19 (Xin et al., 2018). In addition, very weak or no FISH signals were detected in the centromeric regions in most poplar chromosomes (Figure 1), suggesting that the centromeres of most poplar chromosomes may contain exclusively repetitive DNA sequences.

Details are in the caption following the image
Figure 1
Open in figure viewer
PowerPoint
Chromosome painting of chromosome-specific oligo probes on mitotic metaphase cells of Populus trichocarpa.

(a) Chromosome 4 (green) and 5 (red). (b) Chromosome 13 (green) and 9 (red). (c) Chromosome 11 (green) and 12 (red). (d) Chromosome 16 (green) and 15 (red). (e) Chromosome 1 (green) and 17 (red). (f) Chromosome 14 (green) and 7 (red). (g) Chromosome 18 (green) and 2 (red). (h) Chromosome 3 (green) and 19 (red). (i) Chromosome 6 (green) and 10 (red). (j) Chromosome 2 (green) and 8 (red).

Note: the blue fluorescence in situ hybridization signals in (g), (h) and (i) are from the Pt45 probe that labels the positions of poplar centromeres. Bars = 10 ;m.

We then evaluated the quality of the painting probes in hybridization to chromosomes from other poplar species. We selected four poplar species belonging to four different sections in the genus, including Populus tomentosa (section Leuce), Populus deltoides (section Aigeiros), Populus lasiocarpa (section Leucoides) and Populus euphratica (section Turanga). Surprisingly, all 19 painting probes generated highly similar FISH signal patterns on 19 individual chromosomes in all four species (Figure 2). We did not observe cross-chromosome hybridization from any of the 19 probes in the four poplar species, indicating that no cytologically visible structural inter-chromosome rearrangements have occurred among the five poplar species. The arm ratio for each of the 19 chromosomes appeared to be similar among the five species. These results revealed a highly conserved karyotype of these five species that have been diverged for about 14 Myr (Ma et al., 2013; Wang et al., 2014; Ma et al., 2019). We observed particularly weak signals in the centromeres of most P. euphratica chromosomes (Figure 2). This is likely to have been caused by the divergence of centromeric DNA sequences in the two species and/or expansion of centromeric and/or pericentromeric regions in P. euphratica.

Details are in the caption following the image
Figure 2
Open in figure viewer
PowerPoint
Karyotypes of five Populus species.

Chromosomes 1–19 from each species are arranged from left to right. The red fluorescence in situ hybridization signals on the chromosomes were from chromosome-specific oligo probes. The centromeres of the chromosomes are aligned by a dotted line. Bars = 10 ;m.

Comparative FISH mapping of the 5S and 45S rDNA in poplar species
We wondered if the unpainted regions of chromosome 8 and 14 were associated with the 45S ribosomal RNA genes (45S rDNA) since rDNA-related oligos were not included in any painting probes. We conducted FISH mapping of 45S rDNA in all five poplar species and used the chromosome painting probes to assign the 45S rDNA loci to specific chromosomes (Figure 3). Interestingly, the 45S rDNAs were mapped to the distal regions on the short arms of chromosomes 8 and 14 in P. trichocarpa (Figure 3a) as well as in P. deltoides (section Aigeiros) and P. lasiocarpa (section Leucoides). Therefore, the unpainted regions on chromosomes 8 and 14 are associated with 45S rDNA. In P. tremula ; tremuloides and triploid P. tomentosa, which both belong to section Leuce, we detected a single 45S rDNA locus on chromosome 14 (Figure 3c–f). Thus, these species have probably lost the 45S rDNA locus on chromosome 8. Surprisingly, P. euphratica (section Turanga) contained a single 45S rDNA locus located at the distal end of the short arm of chromosome 9 (Figure 3g). In contrast to the variable locations of the 45S rDNA, the 5S rDNA was mapped to chromosome 17 in all five species (Figure 3b,d,f,h).

Details are in the caption following the image
Figure 3
Open in figure viewer
PowerPoint
Fluorescence in situ hybridization (FISH) of 5S rDNA and 45S rDNA in different Populus species.

(a) The FISH mapping of chromosome 8 (red), 14 (green) and a 45S rDNA probe (yellow) on a mitotic metaphase cell of Populus trichocarpa. (b) The FISH mapping of chromosome 17 (red) and a 5S rDNA probe (green) on a mitotic metaphase cell of P. trichocarpa. (c) The FISH mapping of chromosome 17 (red) and 14 (green) on a mitotic metaphase cell of Populus tremula ; tremuloides. (d) The same metaphase cell as in (c) was reprobed with 45S rDNA (green) and 5S rDNA (red). (e) The FISH mapping of chromosome 14 (red) and 17 (green) on a mitotic metaphase cell of triploid Populus tomentosa. (f) The same metaphase cell as in (e) was reprobed with 45S rDNA (green) and 5S rDNA (red). (g) The FISH mapping of chromosome 9 (red) and 45S rDNA (green) on a metaphase cell of Populus euphratica. (h) The FISH mapping of chromosome 17 (red) and 5S rDNA (green) on a metaphase cell of P. euphratica. Bars = 10 ;m.

Comparative karyotyping of P. trichocarpa and P. euphratica
To further reveal the conservation of the karyotype among the poplar species, we conducted detailed comparative karyotyping between P. trichocarpa and P. euphratica. These two species diverged about 14 Myr ago (Ma et al., 2013), and are the most distantly related among the five species used in this study. We first identified all chromosomes in the same metaphase cells from both species and then measured the size of each chromosome. Identification of all P. trichocarpa chromosomes in the same metaphase cell was accomplished by five rounds of sequential FISH mapping using Pt45, 45S rDNA and the painting probes (Figure 4). Three different painting probes (digoxigenin-labeled in red, biotin-labeled in green, and labeled by both digoxigenin and biotin, which results in a yellow hybridization color) were used in the first two rounds of FISH (Figure 4a,d). Five different painting probes (two digoxigenin-labeled, two biotin-labeled and one labeled by both digoxigenin and biotin) were used in the third and fourth rounds of FISH (Figure 4g,j). The two chromosomes labeled with the same color in the third and fourth rounds of FISH can be distinguished by their positions on the centromeres (Figures 1g,h,i and 4h,i,l) or by the 45S rDNA mark (Figures 3a and 4k,m). Pt45 contains a centromere-specific repeat. Thus, FISH results with Pt45 confirmed the position of the centromere in each chromosome. Pt45 and 45S rDNA were used in the last round of FISH (Figure 4m). Chromosomes 1 and 17, respectively the largest and one of the smallest chromosomes, were not painted but can be readily identified (Figure 4n).

Details are in the caption following the image
Figure 4
Open in figure viewer
PowerPoint
Sequential fluorescence in situ hybridization (FISH) to identify all 19 chromosomes in the same metaphase cell of Populus trichocarpa.

(a–c) The first round of FISH using chromosome 5 (red), 4 (green) and 13 (yellow) painting probes. Red (b) and green (c) fluorescence signals were digitally separated from (a). (d–f) Second round of FISH using chromosome 15 (red), 16 (green) and 9 (yellow) painting probes. Red (e) and green (f) fluorescence signals were digitally separated from (d). (g–i) Third round of FISH using chromosome 3 (red), 19 (red), 2 (green), 18 (green) and 11 (yellow) painting probes. Red (h) and green (i) fluorescence signals were digitally separated from (g). (j–l) Fourth round of FISH using chromosome 7 (red), 14 (red), 6 (green), 10 (green) and 12 (yellow) painting probes. Red (k) and green (l) fluorescence signals were digitally separated from (j). (m) Fifth round of FISH using Pt45 (red) and 45S rDNA (green) probes. (n) The FISH signals of all the painting probes and 45S rDNA were merged together. (o) Chromosomes stained with 4;,6-diamidino-2-phenylindole identified based on the FISH results from the painting probes.

The arrows in (h), (i) and (l) indicate the positions of the centromeres, which can be used to distinguish chromosome 3 from 19 (h), 2 from 18 (i), 6 from 10 (l). Bars = 10 ;m.

We analyzed the karyotype of P. euphratica with the same set of oligo probes, but using a different strategy. We used seven rounds of sequential FISH to distinguish all 19 pairs of chromosomes. In each of the first six rounds three different painting probes (digoxigenin-labeled, biotin-labeled and labeled by both digoxigenin and biotin) were used to hybridize to three chromosomes (Figure 5). Pt45 was used in the final round to indicate the positions of the centromeres of all chromosomes (Figure 5g). The measurement was conducted on each chromosome in 10 complete metaphase cells for each species. The karyotypes of the two species were highly similar (Figure 6, Table 1). Only a slight arm ratio difference was observed on a few chromosomes, including chromosomes 1, 3, 8 and 9, between the two species (Table 1). Thus, these two species maintained a highly conserved karyotype after about 14 Myr of divergence.

Details are in the caption following the image
Figure 5
Open in figure viewer
PowerPoint
Sequential fluorescence in situ hybridization (FISH) to identify all 19 chromosomes in the same metaphase cell of Populus euphratica.

(a) First round of FISH using chromosome 4 (green), 5 (red) and 13 (yellow) painting probes. (b) Second round of FISH using chromosome 16 (green), 15 (red) and 9 (yellow) painting probes. (c) Third round of FISH using chromosome 18 (green), 19 (red) and 12 (yellow) painting probes. (d) Fourth round of FISH using chromosome 6 (green), 7 (red) and 1 (yellow) painting probes. (e) Fifth round of FISH using chromosome 2 (green), 3 (red) and 11 (yellow) painting probes. (f) Sixth round of FISH using chromosome 10 (green), 17 (red) and 14 (yellow) painting probes. (g) Seventh round of FISH using the centromeric repeat probe Pt45 (red). (h) Chromosomes stained with 4;,6-diamidino-2-phenylindole identified based on the FISH results from the painting probes. Bars = 10 ;m.

Details are in the caption following the image
Figure 6
Open in figure viewer
PowerPoint
Ideogram of the karyotype of Populus trichocarpa (a) and Populus euphratica (b). Black ellipses indicate the position of the centromere.
Table 1. Relative lengths and arm ratios of mitotic metaphase chromosomes of Populus trichocarpa and Populus euphratica
Chromosomea Relative lengthb (%) Arm ratioc
P. trichocarpa P. euphratica P. trichocarpa P. euphratica
1 11.02 ± 0.83 10.28 ± 0.58 1.74 ± 0.17 1.58 ± 0.15
2 6.63 ± 0.67 6.38 ± 0.43 2.64 ± 0.32 2.73 ± 0.47
3 5.30 ± 0.28 5.27 ± 0.16 2.33 ± 0.29 2.46 ± 0.48
4 5.79 ± 0.46 5.87 ± 0.60 1.18 ± 0.10 1.21 ± 0.13
5 6.05 ± 0.38 6.28 ± 0.43 1.22 ± 0.13 1.24 ± 0.17
6 6.72 ± 0.35 6.94 ± 0.53 1.15 ± 0.11 1.21 ± 0.11
7 4.30 ± 0.26 4.57 ± 0.34 1.21 ± 0.10 1.21 ± 0.12
8d 5.41 ± 0.40 5.57 ± 0.54 3.35 ± 0.40 3.00 ± 0.38
9d 3.70 ± 0.42 3.98 ± 0.24 7.17 ± 0.54 5.88 ± 1.44
10 6.19 ± 0.44 5.67 ± 0.28 3.16 ± 0.23 3.17 ± 0.53
11 4.52 ± 0.46 4.51 ± 0.40 1.19 ± 0.14 1.24 ± 0.08
12 4.24 ± 0.39 4.04 ± 0.48 1.19 ± 0.11 1.20 ± 0.13
13 4.20 ± 0.27 4.54 ± 0.32 1.18 ± 0.08 1.16 ± 0.09
14d 5.11 ± 0.30 4.90 ± 0.33 3.23 ± 0.37 3.30 ± 0.78
15 4.11 ± 0.31 4.43 ± 0.38 1.22 ± 0.11 1.24 ± 0.14
16 3.95 ± 0.45 4.00 ± 0.34 1.28 ± 0.12 1.23 ± 0.11
17 4.22 ± 0.37 4.36 ± 0.64 1.26 ± 0.15 1.18 ± 0.10
18 4.24 ± 0.35 4.31 ± 0.38 1.20 ± 0.09 1.15 ± 0.12
19 4.30 ± 0.53 4.10 ± 0.45 1.21 ± 0.09 1.23 ± 0.16
a Measurement was conducted on each chromosomal arm in 10 metaphase cells. b Relative length = 100 ; chromosome length/total complement length. c Arm ratio = length of the long arm/length of the short arm. d The 45S rDNA on the short arm of chromosomes 8 and 14 in P. trichocarpa and chromosome 9 in P. euphratica were not included in the measurement.
Identification of unanchored sequences associated with the poplar sex chromosome
The unpainted regions on chromosomes 8 and 14 are spanned by the 45S rDNA, thus the unpainted region on chromosome 19, the sex chromosome, is the only cytologically visible region that is not covered by the chromosome painting probes. The current reference genome of P. trichocarpa includes 422.9 Mb of sequences (version 3.0, http://www.phytozome.net/poplar). Approximately 39 Mb of sequences have not been anchored to chromosomes, which may result in the unpainted regions associated with chromosome 19. We attempted to identify and assign some large unanchored sequence scaffolds to chromosomes using FISH.

We selected all unassembled scaffolds with a size >500 kb. We used BLAT software (Kent, 2002) with default parameters to perform a similarity search between unassembled scaffolds and genomic sequences which have been assembled into chromosomes. Then the genomic sequences of unassembled scaffolds that show similarity to assembled sequences were eliminated in the following analysis. We then used RepeatMasker (http://www.repeatmasker.org/) to eliminate repetitive sequences in all unanchored scaffolds. These analyses revealed that the majority of the unanchored scaffolds contained nearly exclusively repetitive DNA sequences. We only identified a total of 42 single-copy DNA sequences from six scaffolds. We selected two single-copy DNA sequences (;3 kb) from scaffolds 22 and 25, respectively, as FISH probes (Table S1). The probe derived from scaffold 22 was mapped to the centromeric region of chromosome 3 (Figure 7a). Interestingly, the probe derived from scaffold 25 was mapped to the distal end of the short arm of chromosome 19 (Figure 7b), which is not covered by the chromosome painting probe. Thus, scaffold 25 will provide a valuable seeding sequence to complete the sequencing of the poplar sex chromosome.

Details are in the caption following the image
Figure 7
Open in figure viewer
PowerPoint
The fluorescence in situ hybridization (FISH) mapping of single-copy sequences associated with two unanchored scaffolds of Populus trichocarpa.

(a) The FISH mapping of a single-copy sequence associated with scaffold 22 (red), a chromosome 3 painting probe (yellow) and the centromeric repeat Pt45 (green) on a metaphase cell of P. trichocarpa.

(b) The FISH mapping of a single-copy sequence associated with scaffold 25 (red) and the chromosome 19 painting probe (green) on a metaphase cell of P. trichocarpa. Bars = 10 ;m.

Arm orientation of chromosomes associated with linkage groups 2 and 8
The 19 pseudomolecules of the current P. trichocarpa reference genome were assembled and coordinated with the DNA markers on the 19 genetic linkage groups constructed in P. trichocarpa. We named each of the 19 P. trichocarpa chromosomes according to the 19 genetic linkage groups (Yin et al., 2004; Tuskan et al., 2006). However, the chromosome painting probes cannot be used to reveal the orientation of the short/long arms of each chromosome with the start (base pair 1) and end of the 19 pseudomolecules. We attempted to resolve the orientation of chromosomes 2 and 8 since the lengths of the two arms associated with these two chromosomes can be readily distinguished (Figure 2).

We designed two single-copy DNA probes from both ends of pseudomolecules 2 and 8 (Table S2). Probes S1 and S2 were designed from the regions at 3684 kb and 22 970 kb on pseudomolecule 2 (Figure 8b). We conducted FISH experiments using these two probes together with a centromere-specific repeat Pt45 (Figure 8a). Probes S1 and S2 were detected on the long and short arms of chromosome 2, respectively (Figure 8a,b). The long arm of chromosome 2 is orientated with the start of pseudomolecule 2. Therefore, the start–end orientation of the current pseudomolecule 2 should be inverted. Similarly, we designed probes S3 and S4 at 6100 and 18 595 kb on pseudomolecule 8 (Figure 8d). We then performed FISH using these two probes together with Pt45 (Figure 8c). Probes S3 and S4 were detected on the long and short arms of chromosome 8, respectively (Figure 8c,d). The short arm of chromosome 8 is orientated with the end of pseudomolecule 8. Therefore, the start–end orientation of the current pseudomolecule 8 should also be inverted.

Details are in the caption following the image
Figure 8
Open in figure viewer
PowerPoint
Fluorescence in situ hybridization (FISH)-based confirmation of the arm orientation associated with chromosomes 2 and 8.

(a) The FISH mapping of probes S1 (green), S2 (red), chromosome 2 painting probe (yellow), and centromeric repeat Pt45 (blue) on a metaphase cell of Populus trichocarpa.

(b) The physical locations of Pt45 and two single-copy gene sequence probes (S1 and S2) on the pseudomolecule of chromosome 2 of P. trichocarpa.

(c) The FISH mapping of probes S3 (green), S4 (red), chromosome 8 painting probe (yellow) and the centromeric repeat Pt45 (blue) on a metaphase cell of P. trichocarpa.

(d) The physical locations of Pt45 and two single-copy gene sequence probes (S3 and S4) on the pseudomolecule of chromosome 8 of P. trichocarpa. All bars = 10 ;m.

Discussion
Chromosome number is the most important component of a karyotype. Dramatic changes in chromosome number have been reported in several animal groups. For example, muntjac deer show major variation of chromosome number, the Indian muntjac (Muntiacus muntjak) having 2n = 6 (female) or 7 (male) and the Chinese muntjac (Muntiacus reevesi) 2n = 46. Remarkably, these two species can produce viable F1 hybrids (2n = 27) in captivity (Shi et al., 1980). Cytogenetic studies have revealed that the changes in chromosome number in muntjac deer were caused by tandem chromosome fusion (Yang et al., 1995; Yang et al., 1997). An even more extensive variation in chromosome number was reported in the insect genus Apiomorpha, an endemic Australian gall-inducing scale insect genus, which exhibits a 48-fold variation in chromosome number with diploid numbers ranging from 4 to about 192 (Cook, 2000). It was hypothesized that chromosomal fusion and/or fission events were responsible for the changes in chromosome number in Apiomorpha (Cook, 2000). Plant species in the Brassicaceae family are known for having highly variable basic chromosome numbers, a result of polyploidization-mediated chromosome repatterning (Liu et al., 2014; Mandakova et al., 2017).

Chromosome number, however, often represents the strongest constraint on karyotype evolution. Closely related species typically maintain an identical or similar chromosome number. In such a situation, chromosome rearrangements, especially inter-chromosomal translocations, are often the major source driving karyotype evolution. For example, gibbons are small arboreal apes that diverged from the great apes approximately 17 Myr ago (Carbone et al., 2014). Although the chromosome numbers, ranging from 38 to 52, of gibbons are similar to those of humans (2n = 46) and the great apes (2n = 48), gibbon genomes show rapid karyotype evolution, based on a number of inter-chromosomal translocations (Jauch et al., 1992; Stanyon et al., 2008; Carbone et al., 2014). We previously observed a very similar karyotype among Solanum species; however, inter-chromosomal translocations were detected in several species (Braz et al., 2018).

The Populus species showed a remarkably conserved karyotype. All 19 chromosomes from five different species, representing the diversity of the genus, have maintained a similar morphology. We did not observe any inter-chromosomal rearrangements (Figure 2). We cannot exclude the possible existence of small inter-chromosomal rearrangements that may not be detectable with the current technique. Such rearrangements, if they exist, are likely to be involved in chromosomal segments of sub-megabase size. The pericentromeric regions of most P. euphratica chromosomes showed faint painting signals (Figure 2), suggesting that the sequences of P. euphratica chromosomes show a relatively higher level of divergence than other Populus species. This result is in agreement with that of section Turanga, which includes P. euphratica and represents the most ecologically idiosyncratic section in Populus (DiFazio et al., 2011). A high level of genetic synteny among the Populus species was previously unveiled by comparative genetic mapping and genome sequencing (Gaudet et al., 2008; Pakull et al., 2009; Paolucci et al., 2010; Ma et al., 2013, 2019; Mousavi et al., 2016; Yang et al., 2017). Comparative linkage mapping has revealed discrepancies in marker order among different Populus species (Gaudet et al., 2008; Paolucci et al., 2010). Although genotyping errors can cause such discrepancies in marker order (Gaudet et al., 2008; Paolucci et al., 2010), chromosomal inversions may have resulted in some of the marker order changes. Potato (Solanum tuberosum) and tomato (Solanum lycopersicum), diverged for about 5–8 Myr, share a highly similar karyotype without inter-chromosomal rearrangements (Braz et al., 2018). However, inversions exist in each of the 12 pairs of potato/tomato chromosomes. These inversions were initially identified by comparative linkage mapping (Tanksley et al., 1992), and were later confirmed by FISH-based cytogenetic mapping (Iovene et al., 2008; Tang et al., 2008) and genome sequencing (The Potato Genome Sequencing Consortium, 2011; The Tomato Genome Consortium, 2012).

There is strong evidence for heterogeneity in the rate of molecular evolution between woody and herbaceous species, which can be attributed to a number of factors including woody versus herbaceous species, annual versus perennial and self-pollinators versus outcrossers (Gaut et al., 1996; Laroche et al., 1997; Kay et al., 2006; Smith and Donoghue, 2008). The molecular clock in Populus was estimated to be at only one-sixth the estimated rate of that of Arabidopsis (Tuskan et al., 2006). Woody species have the potential to successfully contribute gametes to multiple generations. It was proposed that the recurrent contributions of ‘ancient gametes’ from very old individuals may account for the markedly reduced rate of poplar sequence evolution (Tuskan et al., 2006). These previous studies have demonstrated that woody species evolve more slowly than herbaceous species. We hypothesize that woody species are also slower than herbaceous plants to generate and fix rearranged chromosomes or altered chromosome numbers. Karyotyping of multiple species within the same genus has been conducted in several genera of woody plants, including Pinus (Hizume et al., 2002), Nothofagus (Acosta and Premoli, 2018) and Eucalyptus (Ribeiro et al., 2016). Intriguingly, all these studies demonstrated a highly similar karyotype among different species in the same genus. These results support the karyotypes of woody species being more stable than those of herbaceous plants, which is parallel with slower rate of molecular evolution associated with woody plants.

Experimental procedures
Plant materials and chromosome preparation
One triploid poplar species P. tomentosa (2n = 3x = 57) and six diploid Populus species (2n = 2x = 38), including P. tomentosa, P. tremula ; tremuloides, P. deltoides, P. trichocarpa, P. lasiocarpa and P. euphratica were used in FISH mapping. To prepare mitotic metaphase chromosomes, root tips were harvested from tissue culture seedlings and pre-treated with 0.7 mm cycloheximide at room temperature (25°C) for 3 h and fixed in Carnoy’s fixative. Chromosomes were prepared using the stirring method according to published protocols (Xin et al., 2018).

Development of chromosome painting probes and single-copy sequence probes
Chromosome painting probes were developed based on the reference genome of P. trichocarpa (version 3.0, http://www.phytozome.net/poplar) using Chorus2 (https://github.com/zhangtaolab/Chorus2). The design and development of the oligo-based painting probes followed our previous procedures with only minor modifications (Xin et al., 2018). Briefly, the P. trichocarpa pseudomolecules were computationally divided into 45-nt oligos in a step size of 5 nt. Oligos were removed if they could be mapped to two or more locations (with 75% similarity) in the genome. Oligos with Tm – hairpin Tm > 10°C were kept for the pipeline. Finally, P. trichocarpa shotgun sequences from SRA (https://www.ncbi.nlm.nih.gov/sra, SRR2895390, SRR2895392, SRR2895393, SRR2895394, SRR2895395, SRR2895397, SRR2895398) were used for repeat sequence filtering by applying ChorusNGSfilter.py and ChorusNGSselect.py script included in the Chorus2 software. The 19 oligo libraries were synthesized by Arbor Biosciences (https://arborbiosci.com/). The FISH probes based on single-copy gene sequence of chromosome 2, 8 and unassembled scaffolds (length >500 kb) were developed following previously published procedures (Xin et al., 2018).

Oligo-FISH and karyotyping
Probe preparation from the synthesized oligo libraries was conducted as described previously (Han et al., 2015). The biotin- or digoxigenin-labeled single-stranded oligos prepared from the libraries were used as FISH probes. The plasmids containing 5S and 45S rDNA from rice (Oryza sativa L.), a poplar centromeric repeat probe Pt45 and six single-copy DNA sequence probes were also used in FISH. These probes were labeled by nick translation with either digoxigenin-dUTP or biotin-dUTP. Amplification and gel recovery for the single-copy DNA probes followed published protocols (Xin et al., 2018). The primers for the single-copy gene probes are listed in Tables S1 and S2.

Fluorescent in situ hybridization was performed according to published protocols (Braz et al., 2018; Xin et al., 2018) with minor modifications. The hybridization mixture (100 ng of chromosome painting probes, 50% formamide, 10% dextran sulfate, 2; SSC) was applied to denatured chromosomal slides and incubated for 24 h at 37°C. For single-copy DNA FISH, approximately 3500 ng of sheared genomic DNA (with average size of 100 bp) prepared from P. trichocarpa was used as blocking DNA. Biotin-labeled probes and digoxigenin-labeled probes were detected by Alexa Fluor 488 streptavidin and rhodamine anti-digoxigenin, respectively. Chromosomes were stained with 4;,6-diamidino-2-phenylindole (DAPI) in VectaShield antifade solution (Vector Laboratories, https://vectorlabs.com/). The FISH images were captured using a CCD camera attached to an Olympus BX51 fluorescence microscope (https://www.olympus-lifescience.com/). The final image contrast was processed using adobe photoshop 5.0.

High-quality slides were used for repeated FISH. After the first round of FISH and image capture, the slides were washed in 1; PBS for 15 min to remove the coverslip and washed for another 5 min in 1; PBS at 42°C. The slides were then washed twice in 2; SSC at 42°C (15 min each). Finally, the slides were dehydrated in an ethanol series (70%, 90% and 100%, 5 min each), denatured again in 70% formamide at 85°C for 2 min, dehydrated in a second ethanol series and reprobed with different probes.

The short (S) and long (L) arms of individual chromosomes (without including the 45S rDNA region), relative chromosome length and arm ratio were measured and calculated from 10 complete metaphase cells for each poplar species using the ‘measurement’ tool of adobe photoshop.

Acknowledgements
The research was supported by grant 31670603 from the National Natural Science Foundation of China, grant 16KJA220001 from the Jiangsu Provincial Key Basic Research Foundation for Universities, the Doctorate Fellowship Foundation of Nanjing Forestry University and funds from the Priority Academic Program Development of Jiangsu Higher Education Institutions, National Science Foundation grant IOS-1444514 and Michigan State University startup funds to JJ.

Комментарий. Мой. Анализ кариотипа тополя 38 2n-хромосом. Обращено внимание на вариации числа хромосом у щитовок 48 вариациях от 4 до 192, примерно, где летающие формы - только мужские:

alphapedia.ru/w/Apiomorpha

Apiomorpha - это род щитовка, вызывающая галлы у видов Eucalyptus. Галлы инициируются нимфами первой стадии (ползучими животными) при росте нового растения, и, когда они созревают, галлы проявляют выраженный половой диморфизм. Галлы, вызванные самками, являются одними из самых крупных и наиболее впечатляющих из галлов, вызванных членистоногими, в то время как у самцов галлы маленькие и большинство из них трубчатые. Apiomorpha известен только из Австралии и Новой Гвинеи, хотя его хозяин, Eucalyptus, также имеет более широкое распространение в Индонезии.

Apiomorpha в настоящее время классифицируется в Eriococcidae, но это семейство не является монофилетическим. Конец цитирования.

Комментарий. Мой. Продолжение. Спаривание в неволе индийского и китайского мунтжаков даёт в потомстве кариотип 2n из 27 хромосом, где китайский мунтжак имеет количество хромосом 46, раное человеческому. Надо полагать, что только самка китайская и самец индийский дают такое кариотипное число хромосом.

Человек, по всей видимости, так же имеет (исторические, возможно, не вымершие ещё или где-то) определённые виды, скажем с 46 и даже нуль хромосом, где 46 - принадлежность женской сферы, а нуль хромосом - только интерфазный гетерохроматин собственного или транс-волновой фрактальный родословный материал трансперсонального происхождения Божественного Вида. Конечно, не исключены формы скрещивания типа особей разнополого соития между видами с разными количествами гаплоидных наборов хромосом, в итоге дающем стабильное антропоморфное число 46 хромосом, наподобие индийского и китайского мунтжаков, где антропоформные формы парамунтжаков могли быть летающими, неважно, какого пола, ангелами или нимфами.

По сравнению с рассуждениями, где люди не понимают сути антропогенеза, это только первые шаги не осуждения, а объяснения непонятных для человечества жертв и каннибализма.