Run batches with Novoplasty

I was asked to help out with using ‘Novoplasty’ to build complete  chloroplast sequences from shorter dna strands. It’s a pretty amazing app that’s easy enough to use but it only lets you run one seed file at a time – (I’m assuming if you’re reading this you understand what ‘Novoplasty’ is!). Doing lots of runs can quickly eat up your day so I decided to write a perl script that will run batches of seed files through ‘Novoplasty’. Following is how you can use it.

First download a copy of ‘Novoplasty’ and ‘Batch Novoplasty’.

Get ‘NOVOPlasty’ here.

Get ‘Batch Novoplasty’ and ‘Split Fasta File’ here.

The batch script has been written to work with version Novoplasty 2.5.6. You can open the ‘Batch Novoplasty’ in a text editor and change this to work with other versions. This is hard coded near the bottom of the script so be careful when you change the text! Feel free to change the message that’s printed when the script finishes, if it makes your life better!

You can change the version of Novoplasty that the batch script works with by typing over the name near the bottom of the script.

 

Place ‘Novoplasty’ and ‘Batch Novoplasty’ files in the same folder. Open a terminal window, drag ‘Batch Novoplasty’ into the terminal window, and click return. This will create three folders and ask you to place your files in them. The folders are:

  • 1_Seed_Files
  • 2_Reads_Forward
  • 3_Reads_Reverse

Place your .fasta files containing the seed data into the ‘1_Seed_Files’ folder. Each .fasta file should contain one seed, best to clean the data by removing the header and any lower case a, g, c’s or t’s from the beginning.  ‘Novoplasty’ will only read in 100 characters so give it good data to work with.

If your sequences have been supplied in a single .fasta file I’ve attached another perl script that can be used to pull them apart. It relies on each sequence starting with “>Seq_”. The text that follows “>Seq_” is used to create a unique name for each seed file. Either start all your seed fragments with “>Seq_” (followed by a unique identifier like a bar code number) or change the variable inside the script to suit your current format. You will need to name the file containing all your seed fragments “split.fasta”. This script assumes the first 100 characters of your seed fragment are header details and removes them, it also removes any lower case a, g, c’s or t’s from the beginning of the seed.

Rename the seed file to split “split.fasta” and place it in the same folder as the “Split Fasta File” script. Open a terminal window, drag the “Split Fasta File” script in and hit return. The script should spit out a new fasta file for each occurrence of your delimiter string i.e. “>Seq_”.

 

Name the file containing all your seeds “split.fasta” or change the variable at the top of the script. Make sure each new seed sequence inside the split.fasta file starts with “>Seq_” or change the variable in the script to match your delimiter.

 

If running the script gives a permission error, check that the script file is set to be executable. Part of the script that passes the middle 100 characters of your seed to ‘Novoplasty’ has been commented out. The idea was that the middle 100 characters should be clear of any messy sequencing errors that often occur at the beginning and end of a read. If you liked to use this, simply uncomment the section by removing the # at the beginning of each line. Move all the resulting seed files into the ‘1_Seed_Files’ folder for the Batch script to use.

 

Remove the # at the beginning of these lines if you want to use the middle 100 characters.

 

Next place your forward and reverse data into the folders ‘2_Reads_Forward’ and ‘3_Reads_Reverse’. The batch should work with .fastq or . fastq.gz, though I’ve only tested it with fastq.gz files. The script will only read in one file per folder. Jump into terminal again, drag and drop the ‘Batch Novoplasty’ file into your terminal window, hit return and that ‘should’ be it. Your seed files will be read in one at time and built using the supplied forward and reverse files. You won’t get all the normal feed back supplied by ‘Novoplasty’, just a simple ‘processing Seed file 1’ etc…

Note changing  variables in the config file won’t have an effect on ‘Novoplasty’. The batch script recreates the config for every new seed file. If you need different settings in the config file make the changes inside the ‘Batch Novoplasty’ script.

If you want to run multiple batches at the same time, just set up a folder for each batch, put ‘Novoplasty’ and the ‘Batch Novoplasty’ scripts in the same folder and run the batch script in a different terminal window for each batch.

 

Ficus congesta x opposita

Here’s another example of a hybrid arising from two dioecious fig species in my Sydney backyard. The maternal plant (mother) that produced the seed is a Ficus congesta or Red leaf fig that’s commonly encounted in the Australian tropics. The paternal (father) plant that produced the pollen seems to be a Ficus opposita which grows along the east coast of Queensland. From first appearance these species are quite distantly related. F. opposita is a sandpaper fig, while F. congesta has only a slight rough texture to its leaves and a hint of glossiness.

 

The mother plant, Ficus congest foliage
The mother plant, Ficus congesta.

 

Ficus congesta branch
The mother plant, Ficus congesta.

 

Ficus opposita branch
A narrow leaf Ficus opposita. This branch has small unripe fruit, one of the many plants in my backyard that could be the father.

 

Leaf comparison
Left: Ficus opposita, Middle: Ficus congesta x opposita, Right: Ficus congesta. Note the thick strong veins and rough brown hair of the middle leaf  like those of F. opposita. The over all shape and appearance of the leaf is like that of F. congesta.

 

Leaf of Ficus congesta x opposita
Leaf of Ficus congesta x opposita, the leaves are large like those of F. congesta, the vein pattern and leaf shine are very congesta like. The plants growth habit is very F. opposita like.

 

F. congesta can form masses of cauliflorous fruiting stems around the trunk with the fruit staying green and hard before rotting. The trunks of some trees is hidden behind a thick mass of fruiting branches. I don’t know if the fruit ever turns fleshy? Common throughout the rainforest understory and preferring locations with access to water. F. opposita tends to form fruit in pairs towards the ends of its branches that ripen to a fleshy purple-black. It is frequently found in open woodlands, though it too loves water if it can get it.

Both species are placed in the genus “Ficus”, subgenus “Ficus” with F. congesta being grouped with F. hispida and F. septica, while F. opposita is grouped with the sandpaper figs. The two species can occur in sympatry at least in Queensland and there are bound to be naturally formed hybrids. F. congesta is probably able to hybridise with F. hispida and F. septica. This example of crossing with F. opposita suggests that F. congesta could cross with other sandpaper figs as well.

 

Seedling of a plant that appears to be Ficus congesta  x opposita

 

This seedling is about 2 years old and in my eyes displays pretty clear intermediate morphology between the two parent species. The pollinating wasps for both species are not found in Sydney leaving the pollinating wasp of F. coronata as the likely mating intermediate.

 

Left:  unripe Ficus opposita fruit Right: Ficus congesta fruit

 

Aerial roots tighten when they hit the ground.

Sometime ago I read about fig roots tightening as they hit the ground, acting like tent ropes, pulling tight to help hold the tree steady. Claus Mattheck in his book ‘Design in Nature: Learning from Trees’, references a paper by zimmerman et al. saying “the anchored aerial roots tighten themselves up so much that they can easily raise a pot of soil in which they are anchored.” As more roots grow down they cross over each other, welding together to form a network of support for the tree.

 

Fig aerial roots tighten
An aerial root that has pulled tight, and a couple of smaller roots that are still lose and twisted.

 

Recently I noticed some of my plants displaying this behaviour and it made me think about how cleaver it is! How does the root know when it has hit the ground? What makes it decide to start tightening? How does the root go about tightening?

The root may realise it is now in the dark and pull tight, but I’ve seen aerial root tips covered in hessian to keep them moist and they don’t pull tight, so it’s probably not light related. If it is the dark that triggers the root to tighten then the root would need to remember it had previously been in the light, understand that it’s environment had changed and it should now tighten. I really have no idea what mechanism is triggering the root to change it’s behaviour!

Once the root has noticed it is in the ground, how does it then go about tightening the root? It seems to me that the plant would need to kill off cells in the root, like removing links in a loose necklace, causing the root to shorten and therefore tighten. Some how selecting cells that are now excessive and using ‘Apoptosis’ to kill off the cells. What ever mechanism the root uses to tighten it is surely a complicated process that needs to be driven by some intelligence.

Most people still find it hard to believe animals are intelligent, so to expect someone to accept plants are intelligent, is a bit of a stretch. In fact it’s hard to find many biologists or even botanists that accept plants are intelligent so I don’t suggest you bring it up if you want to get a job in science – maybe I should take my own advice!

Anthony Trewavas in his excellent (but complicated to read) book writes about plants using swarm intelligence. Individual parts of the plant communicating and working together as a team. Other references, starting with Darwin, refer to the tips of roots having a section near the tip, that acts like a brain. Hopefully the next generation of biologist will be more open-minded and start to unlock how these amazing processes are driven.

Juvenile and Adult Ficus cerasicarpa leaves

Here’s a side by side comparison of a juvenile leaf from a cultivated seedling of Ficus cerasciarpa (right) and an adult leaf from a cultivated cutting of a wild tree (left). As you can see they are very different, figs having different juvenile foliage is pretty common. Young plants tend to have bigger leaves and internodes, growing as quick as possible to establish themselves while times are good. As the plant matures,  its growth rate, leaves and internodes tend to reduce along with its water and nutrient requirements.

left: Adult ceriscarpa leaf. right: Juvenile ceriscarpa leaf.
left: Adult ceriscarpa leaf.
right: Juvenile ceriscarpa leaf.

 

The juvenile leaf is pretty much  double the size (around 24cm long) of the adult leaf (about 12cm) and it’s unlikely that seedlings in the wild would produce leaves this big. The young leaf is glossy dark green with a fine covering of almost invisible hair on the underside. Both surfaces of the adult leaf are covered in an obvious layer of downy hair, preventing the sheen of the younger leaf. The adult leaf blade is also thicker than that of the juvenile leaf. The vein pattern and colour is similar between the leaves.

 

ceriscarpa adult
Ficus ceriscarpa juvenile, bright green, not much hair.

 

cerasicarpa juvenile
Ficus cerasicarpa adult, dull colouring, tiny hairs can be seen. The brown dots are infections that have occur as the leaf matured.

Ficus tinctoria dispersed by people?

Ficus tinctoria grows throughout the pacific islands and has many uses. The fruit is a staple food for some cultures and can be crushed to produce a dye, hence the common name of ‘Dye Fig’. Other parts of the plant are used for to create fiber and medicines. Its growth habit can vary from a small rambling shrub to a tall lofty tree, growing on rocks, as a strangler or directly in soil.

 

Ficus tinctoria growing as a strangler, Emma Gorge, Western Australia.
Ficus tinctoria growing as a strangler, Emma Gorge, Western Australia.

 

The Australian distribution of Ficus tinctoria is a little unusually. There are two small populations. One on the east coast of Queensland, which I had previously written off as being incorrectly identified as Ficus virgata  which I am yet to see them in the wild or the herbarium. And another population in Western Australia. After recently visiting some of the Western Australian population, it struck me how unusually it was for these trees to be growing where they are. Here is a plant that grows through out the pacific islands and so how did it make its way to the middle of arid Western Australia.

One explanation could be that it is a remnant rainforest tree, which may be true, though I think the area was dried out a long time before the arrival of this plant. My thought is, that Ficus tinctoria was relocated by people. The Western Australian location I saw is a perfect place for people to live, a little oasis in an otherwise dry and hot region and with this plant having so many uses it would make sense for people to move it there.

A 2010 study showed that Livistona palms arrived in Australia about 15,000 years ago and plants from Northern Territory made their way 1000 kilometers to another location in Western Australia, probably with human intervention. Another paper used DNA and Aboriginal stories to recreated the distribution of Baobab trees across Australia, finding that “ancient humans significantly influenced the geographic distribution of Adansonia (Baobab) in northwest Australia”. It would be interesting to see if DNA could reveal where the Australian Ficus tinctoria came from. Were they introduced by seafarers? Are the two Australian populations genetically identical or were the two populations established independently by different groups of people? Have the tinctoria and virgata of Queensland interbreed? So many questions!!!

 

Ficus tinctoria foliage and fruit.
Ficus tinctoria foliage and unripe fruit.

 

Ficus tinctoria foliage.
Ficus tinctoria foliage.

 

Human-mediated introduction of Livistona palms into central Australia: conservation and management implications

New Genetic and Linguistic Analyses Show Ancient Human Influence on Baobab Evolution and Distribution in Australia

 

Adenosperma Population Variation

It’s not unusual for populations separated by vast distances to show variation in form, after all this is how we believe many new species originate. However the variation between two populations of Ficus adenosperma, one in Eastern Qld and the other in Litchfield NT, suggest there is something more than mere geographic isolation at play. Berg and Corner (2005) described adenosperma as growing along the north-eastern coast of QLD and islands to the north of Australia, Solomon Islands, New Hebrides, Moluccas and New Guinea. The description noted variation in the amount of hair on the leaves, the Qld population being mostly hairless, while other populations are distinctly hairy. No where in the description is it mentioned that this species has hollow stems, something I would think is an important identifying feature, nor is it mentioned that adenosperma grows in the Northern Territory.

 

Distribution of adenosperma.
Distribution of adenosperma. Dotted line Berg and Corner 2005, Solid Line area includes another group of adenosperma collections.

 

The accepted distribution of Australian adenopserma includes Litchfield National Park and the north-eastern tip of NT, which may be valid records though something has me wondering if they’re correctly identified. Dixon (2011) in Flora of the Darwin Region mentions that adenosperma rarely occurs in Darwin and has ‘hollow’ stems. As far as I am aware having hollow stems is not a variation, either a species has hollow stems or it doesn’t. All the species of hollow stemmed figs that grow in Australia to my knowledge are consistently hollow.

 

Illustration of NT adenosperma by M. Osterkamp, D.J. Dixon 2011 - Flora of the Darwin Region Volume 1, Moraceae
Illustration of NT adenosperma by M. Osterkamp, D.J. Dixon 2011 – Flora of the Darwin Region Volume 1, Moraceae

 

 

The Sydney Botanical Gardens collection of adensperma is only small, there were about 5 Australian collections with only one from Litchfield. From first impressions this single collection is very different to the QLD specimens, having broad leaves with distinct raised yellow venation and hollow stems. The leaves of QLD adenosperma are generally narrow, more linear than broad and have solid stems. Hollow stems in one population but not in another leads me think of two possible explanations. 1. The Northern Territory plants are not the same species as the QLD plants or 2. The NT plants have mixed parentage, maybe one of the parents is a hollow stemmed species like hispida –  though I doubt hispida is a close enough relative to be able to hybridise with adenosperma. If the NT plants turn out to be a different species that raises the question, which of the populations are truly adenosperma?

 

Fairly typical of QLD collection of adenosperma, showing narrow leaves. Sydney Botanical Gardens Collection ID NSW 819598
Fairly typical of QLD collection of adenosperma, showing narrow leaves and thin stems. Sydney Botanical Gardens Collection ID NSW 819598

 

Litchfield NT collection of adenosperma looking a bit like septica or congesta, Sydney Botanical Gardens Collection ID NSW 819600
Litchfield NT collection of adenosperma looking a bit like septica?, Sydney Botanical Gardens Collection ID NSW 819600

 

The plants in QLD were identified by Berg and or Corner, with an extensive history working with the Figs of Asia, it would seem that the QLD population are correctly identified and the NT plants are incorrectly named. Yet I’m not totally comfortable with that idea. The few adenosperma I saw collected from outside of Australia, appeared to be more like the NT plants, having broader leaves and at least one collection having hollow stems. The illustrations in Berg and Corner also seem more like the NT populations and it is noted that the QLD population are at the extreme end of the variation. So are hollow stems a typical feature of adensperma and the QLD plants are wrongly included in this species, did Berg and Corner somehow forgot to mention this species has hollow stems? Or are the hollow stemmed plants from outside of Australia also potential hybrids with a hollow stemmed species?

 

Hollow stemmed adenosperma collected in New Guinea, Sydney Botanical Gardens Collection ID NSW 819622
Hollow stemmed adenosperma collected in New Guinea, Sydney Botanical Gardens Collection ID NSW 819622

 

Trying to show the hollow stem of this adenosperma collected in New Guinea, Sydney Botanical Gardens Collection ID NSW 819622
Trying to show the hollow stem of this adenosperma collected in New Guinea, Sydney Botanical Gardens Collection ID NSW 819622

 

Only a month ago I was in Litchfield and high on my list was to find some of these ‘adenosperma’, sadly I ran out of time and missed an opportunity to see living plants, which are much easier to identify than dried collections. Hopefully in the future I can see all the collections of the species and come to a clearer understanding of what is going on!

 

Berg and Corner 2005 – Flora Malesiana Volume 17 / Part 2, Series 1 – Seed Plants P 353 – 355

D.J. Dixon 2011 – Flora of the Darwin Region Volume 1, Moraceae

 

 

 

Ripe fruit on exotic fig!

As ‘everyone’ knows, figs are apparently pollinated by a single species of wasp. Therefore a fig species that is moved to new country without its pollinator wasp, shouldn’t form ripe fruit. Well here’s yet another example of an exotic fig, growing in Australia and producing ripe fruit without access to its pollinator.

 

The unripe fruit with its large green spots looks totally different to microcarpa fruit.

 

The plant at hand is a bit of a botanic oddity. Sometimes called Ficus microcarpa var crassifolia and sold under the common names of ‘Green Island’, ‘Green Mound’, ‘Wax Fig’  etc. Small growing to maybe 1.5m – maybe becoming a tree over time, with a distinctly thick round leaf . This plant is widely grown as a shrub or hedge in tropical areas, yet the plant seems to be unrecognised botanically? Berg and Corner in ‘Flora Malesiana’ have synonymised the name of microcarpa var crassifolia with the species and without researching too hard, I haven’t been able to find a true botanically reference to this plant. This may be because the plant has been developed in cultivation and botanists only really care about naturally occurring populations of plants?

It would be interesting to know the origin of this plant. Possibly described  for the first time in ‘Ficus microcarpa var. crassifolia W.C. Shieh in Quart. J. Taiwan Mus. 16: t. 5. 1963’ and mentioned again in ‘Ficus microcarpa var. crassifolia (W.C. Shieh) J.C. Liao in Ser. Publ. Forest. Exp. Forest NTU 62: 79. 1974’. But hey the name of the plant isn’t really the point here and I’ll look into that another day! I’m not even sure this plant is a variety of microcarpa, it looks way too different for my liking, but again, that’s not the focus here.

 

Green Island fig ripe fruit
Green Island fig ripe fruit

 

Green Island fig ripe fruit
Green Island fig ripe fruit – I forgot to get photo’s of the fruit on the plant, so I could be making this up!

 

I’ve had these plants in my collection for over a decade. Each year they happily grow masses of fruit and every year that fruit goes unpollinated. Until this year! One lonely fruit turned dark purple and soften up. It’s now Autumn in Australia, the days are still warm and the nights are rapidly cooling so I’m hoping it will be warm enough to  germinate the seed. It will be interesting to see what the offspring look like. Hopefully they’ll give some clues to which species is the father.

 

Ficus microcarpa, cv Hilli, Green Island
Left, There large rounded leaf of a wild Ficus microcarpa, middle the now unrecognised plant previously refered to as var Hillii probably best called cv Hillii?, and right ‘Green Island’ fig. Note all three plants were in fruit when this was written.

 

Green Island fig
Same order as the above photo with unripe fruit.

 

 

Early sexual maturity, from hybrid vigor?

When growing fruit trees from seed you can be in for a painfully long wait before getting your reward. From the many hundreds of fig seedlings I’ve grown, only a handful have reached sexual maturity. This small sample size has shown that sandpaper figs mature the quickest, some setting fruit around year five. While rock and strangler figs take longer to fruit at around their tenth year.

Many of my sandpaper figs have produced fruit within their first year, which didn’t strike me as being odd. It wasn’t until one of my rock fig seedlings produced fruit in its first year that I clicked to the fact that all these plants are (potential) hybrids. This early fruit production seems to be the result of hybrid vigor or ‘Heterosis’.

 

An odd rock fig seedling with barely twenty leaves is already producing fruit!
An odd rock fig seedling with barely twenty leaves is already producing fruit!

 

Heterosis is a strange phenomenon where the offspring of a mating will produce features that surpass those of both the parents. It tends to happen when distantly related individuals are mated, either from different populations, species or even genus. In plants hybrid vigour often displays as faster or larger growth than the parents exhibited. The unusually early sexual maturity of these cross species Ficus seedlings may be the result of hybrid vigor.

 

rock fig hybrid2
The same plant as above showing young fruit spikes similar to those of Ficus platypoda.

 

rock fig hybrid3
The fruit is small at less than 1cm across with a long thin peduncle, a bit like small cerasicarpa or a long stemmed lilliputiana fruit. I’m yet to see if the fruit gets pollinated.

Real Cherry like cerasicarpa fruit.

The fruit of cerasicarpa is meant to look like that of a cherry, with a long stem leading to a small rounded fruit. However the fruit of the plants I came across in the wild didn’t fit the description. The ‘type’ sample does however have obviously long stemmed fruit and it made me wonder if the stem length varied season to season depending on how good the conditions were?

cerasicarpa holatype Dale Dixon from A Chequered History: the Taxonomy of Ficus platypoda and F. leucotricha (Moraceae: Urostigma sect. Malvanthera) Unravelled
cerasicarpa holatype Dale Dixon from A Chequered History: the Taxonomy of Ficus platypoda and F. leucotricha (Moraceae: Urostigma sect. Malvanthera) Unravelled – note the long thin peduncles.

 

It seems this is the case. The fruit collect from plants in wild had short peduncles (fruit stems) in relation to the size of the fruit. See below.

 

cerasicarpa wild fruit
cerasicarpa wild fruit

 

Cuttings taken from these same wild plants have since produced fruit. From the picture below you can see their peduncles are twice the length of those on fruit collected from the parent plant while growing in the wild. This shows the same plant can produce dramatically different looking fruit depending on the conditions it’s growing in.

 

cerasicarpa unripe Sydney
cerasicarpa cultivated fruit – with long fruit stems

 

The ‘seed’ from these unripe cerasicarpa fruit has been planted even though the fruit hadn’t ripened. Past experience shows that Ficus fraseri fruit doesn’t need to be ripe for there to be some variable seed. It may be possible that other species too will produce seed in unripe fruit. This makes me wonder if there needs to be a certain number of pollinated flowers within a fruit before the fruit will ripen and become soft. I’ve heard that Ficus carica fruit crack when ‘too many’ flowers have been pollinated and each expands and cracks the main fruiting body? Not sure if that’s true…

If the seed from this unripe cerasicarpa fruit does grow then it has probably been pollinated by a local Sydney fig and the resulting seedlings are potentially hybrids.

 

 

Ficus triradiata hybrid – Part 2

In December 2015 I planted seed from a Ficus triradiata growing in my backyard. Though thousands of miles from its pollinating wasp, somehow this tree had set ripe fruit. Three months later the 40cm tall seedlings are old enough to start morphological comparisons with their mother.

tri x top mum left
Left typical triradiata leaf, right the triradiata cross rubiginosa? Top Surface.

 

If I didn’t know better I’d say these seedlings were Ficus rubiginosa and the triradata flowers may well have been fertilised by a rubiginosa. Having a ‘rubiginosa’ like appearance continues to make me think many plants identified as rubiginosa are in fact hybrids.

The plants are in their juvenile stage and this could explain the differences in morphology. The leaves of the seedings have prominent  hydathodes – small white spots on the upper surface of the leaf blade. These are common in many species like rubiginosa and the presence or lack of hydathodes is often used as a species delimiter. The mother plant has no hydathodes. My feeling is that hydathodes would persist throughout the plants life and not just as a stage of maturity, though I have no evidence for this.  The leaves are also darker in colour, more rounded and glossy than a typical triradiata leaf.

On the underside of the leaves the veins are obviously raised and coloured differently to the rest of the lamina. Two strong basal veins form a ‘v’ shape at the base of the mid rib. Triradata’s veins are flat to the touch, more delicate in structure and blend in with the rest of the leaf surface, like those of obliqua.

  • Update: Since writing this I thought I’d actually check my other triradiata plants and it seems they too have hydathodes, at least on the juvenile foliage. This trait alone won’t prove these plants are hybrids.
Left typical triradiata leaf, right the triradiata cross rubiginosa? Bottom Surface.
Left typical triradiata leaf, right the triradiata cross rubiginosa? Bottom Surface.

 

Triradiata cross rubiginosa? Top surface.
Triradiata cross rubiginosa? Top surface.

 

Triradiata cross rubiginosa? Bottom surface.
Triradiata cross rubiginosa? Bottom surface.

 

Typical triradiata, Top surface.
Typical triradiata, Top surface.

 

Typical triradiata, Bottom surface.
Typical triradiata, Bottom surface.